Polynucleotides encoding S99T interferon gamma polypeptide variants and means of expression

ABSTRACT

The present invention relates to novel interferon gamma polypeptide variants having interferon gamma (IFNG) activity, methods for their preparation, pharmaceutical compositions comprising the polypeptide variants and their use in the treatment of diseases, in particular for the treatment of interstitial pulmonary diseases, such as idiopathic pulmonary fibrosis. These novel polypeptide variants all comprise the substitution S99T as compared to the amino acid sequence of huIFNG or fragments thereof. By performing this mutation the naturally occurring N-glycosylation site present at position 97 is significantly better utilized. Preferably, the variants comprise further modifications, e.g. in order to increase the AUC of such variants when administered subcutaneously.

FIELD OF THE INVENTION

The present invention relates to novel interferon gamma polypeptidevariants having interferon gamma (IFNG) activity, methods for theirpreparation, pharmaceutical compositions comprising the polypeptidevariants and their use in the treatment of diseases, in particular forthe treatment of interstitial pulmonary diseases, such as idiopathicpulmonary fibrosis.

BACKGROUND OF THE INVENTION

Interferon gamma (IFNG) is a cytokine produced by T-lymphocytes andnatural killer cells and exists as a homodimer of two noncovalentlybound polypeptide subunits. The mature form of each dimer comprises 143amino acid residues (shown in SEQ ID NO:17), the precursor form thereofincludes 166 amino acid residues (shown in SEQ ID NO:18).

Each subunit has two potential N-glycosylation sites (Aggarwal et al.,Human Cytokines, Blackwell Scientific Publications, 1992) at positions25 and 97. Depending on the degree of glycosylation the molecular weightof IFNG in dimer form is 34-50 kDa (Farrar et al., Ann. Rev. Immunol,1993, 11:571-611).

The primary sequence of wild-type human IFNG (huIFNG) was reported byGray et al. (Nature 298:859-863, 1982), Taya et al. (EMBO J. 1:953-958,1982), Devos et al. (Nucleic Acids Res. 10:2487-2501, 1982) andRinderknecht et al. (J. Biol. Chem. 259:6790-6797, 1984), and in EP 0077 670, EP 0 089 676 and EP 0 110 044. The 3D structure of huIFNG wasreported by Ealick et al. (Science 252:698-702, 1991).

Various naturally-occurring or mutated forms of the IFNG subunitpolypeptides have been reported, including one comprising a Cys-Tyr-CysN-terminal amino acid sequence (positions (−3)-(−1) relative to SEQ IDNO:17), one comprising an N-terminal methionine (position −1 relative toSEQ ID NO:17), and various C-terminally truncated forms comprising127-134 amino acid residues. It is known that 1-15 amino acid residuesmay be deleted from the C-terminus without abolishing IFNG activity ofthe molecule. Furthermore, heterogenecity of the huIFNG C-terminus wasdescribed by Pan et al. (Eur. J. Biochem. 166:145-149, 1987).

HuIFNG muteins were reported by Slodowski et al. (Eur. J. Biochem. 202:1133-1140, 1991), Luk et al. (J. Biol. Chem. 265:13314-13319, 1990),Seelig et al., (Biochemistry 27:1981-1987, 1988), Trousdale et al.(Invest. Ophthalmol. Vis. Sci. 26:1244-1251, 1985), and in EP 146354. Anatural huIFNG variant was reported by Nishi et al. (J. Biochem.97:153-159, 1985).

U.S. Pat. No. 6,046,034 discloses thermostable recombinant huIFNG(rhuIFNG) variants having incorporated up to 4 pairs of cysteineresidues to enable disulphide bridge formation and thus stabilization ofthe IFNG variant in homodimer form.

WO 92/08737 discloses IFNG variants comprising an added methionine inthe N-terminal end of the full (residues 1-143) or partial (residues1-132) amino acid sequence of wild-type human IFNG. EP 0 219 781discloses partial huIFNG sequences comprising amino acid residues 3-124(of SEQ ID NO:17). U.S. Pat. No. 4,832,959 discloses partial huIFNGsequences comprising residues 1-127, 5-146 and 5-127 of an amino acidsequence that compared to SEQ ID NO:17 has three additional N-terminalamino acid residues (Cys-Tyr-Cys). U.S. Pat. No. 5,004,689 discloses aDNA sequence encoding huIFNG without the 3 N-terminal amino acidresidues (Cys-Tyr-Cys) and its expression in E. coli. EP 0 446 582discloses E. coli produced rhuIFNG free of an N-terminal methionine.U.S. Pat. No. 6,120,762 discloses a peptide fragment of huIFNGcomprising residues 95-134 thereof (relative to SEQ ID NO:18).

High level expression of rhuIFNG was reported by Wang et al. (Sci. Sin.B 24:1076-1084, 1994).

Glycosylation variation in rhuIFNG has been reported by Curling et al.(Biochem. J. 272 :333-337, 1990) and Hooker et al., (J. of Interferonand Cytokine Research, 1998, 18: 287-295).

Polymer-modification of rhuIFNG was reported by Kita et al. (Drug Des.Deliv. 6:157-167, 1990), and in EP 236987 and U.S. Pat. No. 5,109,120.

WO 92/22310 discloses asialoglycoprotein conjugate derivatives ofinterferons, inter alia huIFNG.

IFNG fusion proteins have been described. For instance, EP 0 237 019discloses a single chain polypeptide having region exhibiting interferonβ activity and one region exhibiting IFNG activity.

EP 0 158 198 discloses a single chain polypeptide having a regionexhibiting IFNG activity and a region exhibiting IL-2 activity. Severalreferences described single chain dimeric IFNG proteins, e.g. Landar etal. (J. Mol. Biol., 2000, 299:169-179).

WO 99/02710 discloses single chain polypeptides, one example among manybeing IFNG.

WO 99/03887 discloses PEGylated variants of polypeptides belonging tothe growth hormone superfamily, wherein a non-essential amino acidresidue located in a specified region of the polypeptide has beenreplaced by a cysteine residue. IFNG is mentioned as one example of amember of the growth hormone super family, but modification thereof isnot discussed in any detail.

IFNG has been suggested for treatment of interstitial lung diseases(also known as Interstitial Pulmonary Fibrosis (IPF) (Ziesche et al. (N.Engl. J. Med. 341:1264-1269, 1999 and Chest 110:Suppl:25S, 1996) and EP0 795 332) for which purpose IFNG can be used in combination withprednisolone. In addition to IPF, granulomatous diseases (Bolinger etal, Clinical Pharmacy, 1992, 11:834-850), certain mycobacterialinfections (N. Engl. J. Med. 330:1348-1355, 1994), kidney cancer (J.Urol. 152:841-845, 1994), osteopetrosis (N. Engl. J. Med. 332:1594-1599,1995), scleroderma (J. Rheumatol. 23:654-658, 1996), hepatitis B(Hepatogastroenterology 45:2282-2294, 1998), hepatitis C (Int. Hepatol.Communic. 6:264-273, 1997), septic shock (Nature Medicine 3:678-681,1997), and rheumatoid arthritis may be treated with IFNG.

As a pharmaceutical compound rhuIFNG is used with a certain success,above all, against some viral infections and tumors. rhuIFNG is usuallyapplicable via parenteral, preferably via subcutaneous, injection.Maximum serum concentrations have been found after seven hours. Thehalf-life in plasma is 30 minutes after iv administration. For thisreason efficient treatment with rhuIFNG involves frequent injections.The main adverse effects consist of fever, chills, sweating, headache,myalgia and drowsiness. These effects are associated with injectingrhuIFNG and are observed within the first hours after injection. Rareside effects are local pain and erythema, elevation of liver enzymes,reversible granulo- and thrombopenia and cardiotoxicity.

WO 01/36001 discloses novel IFNG conjugates comprising a non-polypeptidemoiety attached to an IFNG polypeptide which have been modified byintroduction and/or deletion of attachment sites for suchnon-polypeptide moieties, e.g. PEG and glycosylation sites.

It is well known that when N-glycosylated molecules, such as IFNG, areproduced in a glycosylating host not all potential N-glycosylation sitesare fully utilized. This means that quite often a mixture of proteinshaving a varying degree of in vivo N-glycosylation is obtained, which inturn has the consequence that subsequent purification is necessary.Furthermore, it is often time-consuming and cumbersome to separateidentical proteins having a varying degree of glycosylation. It has nowsurprisingly been found that by substitution of one or more amino acidresidues located close to an in vivo N-glycosylation site (independentlyof whether said in vivo N-glycosylation site is naturally occurring inIFNG or whether the in vivo N-glycosylation site has been introduced,such as described in WO 01/36001) it is possible to obtain an increasedfraction of fully glycosylated IFNG molecules. In particular, it hasbeen found that changing the naturally occurring N-glycosylation siteN-Y-S at positions 97, 98 and 99 of hIFNG to N-Y-T gives rise to adramatically increased fraction of fully glycosylated IFNG molecules.

BRIEF DISCLOSURE OF THE INVENTION

Thus, in a first aspect the present invention relates to an interferongamma (IFNG) polypeptide variant exhibiting IFNG activity and having theamino acid sequence shown in SEQ ID NO:1 ([S99T]huIFNG), or a fragmentthereof exhibiting IFNG activity.

In a further aspect the present invention relates to a variant of SEQ IDNO:1, including variant of fragments of SEQ ID NO:1 (such as SEQ IDNOS:2-16), wherein said variant comprises at least one furthermodification and exhibits IFNG activity.

In still further aspects the present invention relates to a nucleotidesequence encoding a polypeptide variant of the invention.

In even further aspects the present invention relates to an expressionvector comprising a nucleotide sequence of the invention, and to aglycosylating host cell comprising a nucleotide sequence of theinvention or an expression vector of the invention.

The present invention also relates to a pharmaceutical compositioncomprising a polypeptide variant of invention, to a polypeptide variantof the invention or to a pharmaceutical composition of the invention foruse as a medicament.

Even further aspects of the present invention relates to the use of apolypeptide variant of the invention or to the use of a pharmaceuticalcomposition of invention for the manufacture of a medicament fortreatment of interstitial lung diseases.

Analogously, the present invention also relates to a method for treatingor preventing interstitial lung diseases, said method comprisingadministering to a mammal, in particular a human being, in need thereofan effective amount of a polypeptide variant of the invention or apharmaceutical composition of the invention.

A still further aspect of the invention relates to a population of IFNGpolypeptide variants, or a composition comprising a population of IFNGpolypeptide variants, wherein said population comprises at least 70% ofthe IFNG polypeptide variant of the invention.

In another aspect the present invention relates to a method ofincreasing the degree of in vivo N-glycosylation of a parent IFNGpolypeptide that comprises at least one in vivo N-glycosylation sitewith the amino acid sequence N-X-S, wherein X is any amino acid residueexcept proline, said method comprising substituting the serine residuein said N-X-S amino acid sequence with a threonine residue to obtain anIFNG variant.

In still another aspect the present invention relates to a method forproducing an IFNG polypeptide variant of the invention, said methodcomprising

-   -   (a) culturing a glycosylating host cell comprising a nucleotide        sequence which encodes an IFNG polypeptide variant of the        invention under conditions conducive for expression of the        polypeptide variant;    -   (b) optionally reacting said polypeptide variant with a        non-polypeptide moiety in vitro under conditions conducive for        the conjugation to take place; and    -   (c) recovering the polypeptide variant.

Other aspects of the present invention will be apparent from the belowdescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Western blot of optimised glycosylation variants of rhuIFNG.Left side Western blot: Lane 1: standard, Lane 2: Actimmune®, Lane 3:rhuIFNG, Lane 4: [E38N]rhuIFNG. Middle Western blot: Lane 1: standard,Lane 2: rhuIFNG, Lane 3: [E38N+S40T]rhuIFNG. Right side Western blot:Lane 1: standard, Lane 2: rhuIFNG, Lane 3: [S99T]rhuIFNG, Lane 4:[E38N+S40T+S99T]rhuIFNG.

FIG. 2 shows the IFNG activity in serum-time curve after subcutaneousadministration in rats. •: Actimmune®, :rhuIFNG,:[E38N+S40T+S99T]rhuIFNG.

The same dose was administered for all compounds (1.15×10⁷ AU/kg).

FIG. 3 shows the IFNG activity in serum-time curve after subcutaneousadministration in rats. •: [N16C+S99T]rhuIFNG (5 kDa mPEG attached),:[N16C+S99T]rhuIFNG (10 kDa mPEG attached), :[E38N+S40T+S99T]rhuIFNG.

The [E38N+S40T+S99T] variant was administered in a dose of 1.15×10⁷AU/kg, whereas the two PEGylated variants were administered in a dose of4.6×10⁶ AU/kg.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “conjugate” (or interchangeably “conjugated polypeptide” or“conjugated variant”) is intended to indicate a heterogeneous (in thesense of composite or chimeric) molecule formed by the covalentattachment of one or more polypeptide variant(s) to one or morenon-polypeptide moieties. The term covalent attachment means that thepolypeptide variant and the non-polypeptide moiety are either directlycovalently joined to one another, or else are indirectly covalentlyjoined to one another through an intervening moiety or moieties, such asa bridge, spacer, or linkage moiety or moieties. Preferably, aconjugated polypeptide variant is soluble at relevant concentrations andconditions, i.e. soluble in physiological fluids such as blood. Examplesof conjugated polypeptide variants of the invention include glycosylatedand/or PEGylated polypeptide variants. The term “non-conjugatedpolypeptide variant” may be used about the polypeptide part of theconjugated polypeptide variant.

The term “non-polypeptide moiety” is intended to indicate a moleculethat is capable of conjugating to an attachment group of the IFNGpolypeptide variant. Preferred examples of such molecules includepolymer molecules, lipophilic compounds, sugar moieties or organicderivatizing agents. It will be understood that the non-polypeptidemoiety is linked to the polypeptide through an attachment group of thepolypeptide variant. Except where the number of non-polypeptidemoieties, such as polymer molecule(s), attached to the IFNG polypeptidevariant is expressly indicated every reference to “a non-polypeptidemoiety” attached to the IFNG polypeptide variant or otherwise used inthe present invention shall be a reference to one or morenon-polypeptide moieties attached to the IFNG polypeptide variant.

The term “polymer molecule” is defined as a molecule formed by covalentlinkage of two or more monomers, wherein none of the monomers is anamino acid residue. The term “polymer” may be used interchangeably withthe term “polymer molecule”.

The term “sugar moiety” is intended to indicate a carbohydrate moleculeattached by in vivo or in vitro glycosylation, such as N- orO-glycosylation.

An “N-glycosylation site” has the sequence N-X-S/T/C”, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine. An “O-glycosylation site” is the OH-group of aserine or threonine residue.

The term “attachment group” is intended to indicate an amino acidresidue group capable of coupling to the relevant non-polypeptide moietysuch as a polymer molecule or a sugar moiety. Useful attachment groupsand their matching non-polypeptide moieties are apparent from the tablebelow. Conjugation Attachment Examples of non- method/activated groupAmino acid polypeptide moiety PEG Reference —NH₂ N-terminal, LysPolymer, e.g. PEG mPEG-SPA Shearwater Inc. Tresylated Delgado et al,mPEG critical reviews in Therapeutic Drug Carrier Systems 9(3, 4):249-304 (1992) —COOH C-term, Asp, Glu Polymer, e.g. PEG mPEG-HzShearwater Inc Sugar moiety In vitro coupling —SH Cys Polymer, e.g. PEG,PEG- Shearwater Inc Sugar moiety vinylsulphone Delgado et al,PEG-maleimide critical reviews In vitro coupling in Therapeutic DrugCarrier Systems 9(3, 4): 249-304 (1992) —OH Ser, Thr, OH—, Sugar moietyIn vivo O-linked Lys glycosylation —CONH₂ Asn as part of an Sugar moietyIn vivo N-glycosylation glycosylation site Aromatic Phe, Tyr, Trp Sugarmoiety In vitro coupling residue —CONH₂ Gln Sugar moiety In vitrocoupling Yan and Wold, Biochemistry, 1984, Jul 31; 23(16): 3759-65Aldehyde Oxidized Polymer, e.g. PEG, PEGylation Andresz et al., Ketonecarbohydrate PEG-hydrazide 1978, Makromol. Chem. 179: 301; WO 92/16555,WO 00/23114 Guanidino Arg Sugar moiety In vitro coupling Lundblad andNoyes, Chimical Reagents for Protein Modification, CRC Press Inc. BocaRaton, FL Imidazole ring His Sugar moiety In vitro coupling As forguanidine

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting anN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by N-glycosylation, the term “amino acidresidue comprising an attachment group for the non-polypeptide moiety”as used in connection with alterations of the amino acid sequence of theIFNG polypeptide is to be understood as one, two or all of the aminoacid residues constituting an N-glycosylation site is/are to be alteredin such a manner that either a functional N-glycosylation site isintroduced into the amino acid sequence, removed from said sequence or afunctional N-glycosylation site is retained in the amino acid sequence(e.g. by substituting a serine residue, which already constitutes partof an N-glycosylation site, with a threonine residue and vice versa).

In the present application, amino acid names and atom names (e.g. CA,CB, CD, CG, SG, NZ, N, O, C, etc) are used as defined by the ProteinDataBank (PDB) (www.pdb.org) which are based on the IUPAC nomenclature(IUPAC Nomenclature and Symbolism for Amino Acids and Peptides (residuenames, atom names etc.), Eur. J. Biochem., 138, 9-37 (1984) togetherwith their corrections in Eur. J. Biochem., 152, 1 (1985). CA issometimes referred to as Cα, CB as Cβ. The term “amino acid residue” isintended to indicate an amino acid residue contained in the groupconsisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid(Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine(Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys orK), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N),proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine(Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp orW), and tyrosine (Tyr or Y) residues.

Numbering of amino acid residues in this document is from the N-terminusof [S99T]huIFNG without signal peptide (i.e. SEQ ID NO:1) or, whererelevant, from the N-terminus of huIFNG without signal peptide (i.e. SEQID NO:17).

The terminology used for identifying amino acid positions/substitutionsis illustrated as follows: G 18 indicates position 18 occupied byglycine in the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:17.G18N indicates that the Gly residue of position 18 has been replacedwith an Asn. Multiple substitutions are indicated with a “+”, e.g.G18N+S20T means an amino acid sequence which comprises a substitution ofthe Gly residue in position 18 with an Asn and a substitution of the Serresidue in position 20 with Thr. Alternative substitutions are indicatedwith a “/”. For example, G18S/T covers the following individualsubstitutions: G18S and G18T. Deletions are indicated by an asterix. Forexample, G18* indicates that the Gly residue in position 18 has beendeleted. Insertions are indicated the following way: Insertion of anadditional Ser residue after the Gly residue located at position 18 isindicated as G18GS. Combined substitutions and insertions are indicatedin the following way: Substitution of the Gly residue at position 18with an Ser residue and insertion of an Ala residue after the position18 amino acid residue is indicated as G18SA.

The term “nucleotide sequence” is intended to indicate a consecutivestretch of two or more nucleotide molecules. The nucleotide sequence maybe of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

The term “polymerase chain reaction” or “PCR” generally refers to amethod for amplification of a desired nucleotide sequence in vitro, asdescribed, for example, in U.S. Pat. No. 4,683,195. In general, the PCRmethod involves repeated cycles of primer extension synthesis, usingoligonucleotide primers capable of hybridising preferentially to atemplate nucleic acid.

“Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.

“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

“Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, the nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence for a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide: a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the nucleotidesequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used, inconjunction with standard recombinant DNA methods.

The term “modification”, as used herein, covers substitution, insertionand deletion.

The terms “mutation” and “substitution” are used interchangeably herein.

The term “introduce” is primarily intended to mean substitution of anexisting amino acid residue, but may also mean insertion of anadditional amino acid residue.

The term “remove” is primarily intended to mean substitution of theamino acid residue to be removed for another amino acid residue, but mayalso mean deletion (without substitution) of the amino acid residue tobe removed.

The term “amino acid residue comprising an attachment group for thenon-polypeptide moiety” is intended to indicate that the amino acidresidue is one to which the non-polypeptide moiety binds (in the case ofan introduced amino acid residue) or would have bound (in the case of aremoved amino acid residue).

The term “one difference” or “differs from” as used in connection withspecific modifications is intended to allow for additional differencesbeing present apart from the specified amino acid difference. Thus, inaddition to amino acid residue alterations disclosed herein aiming atoptimising the utilization of glycosylation sites or removing and/orintroducing amino acid residues comprising an attachment group for anon-polypeptide moiety, the IFNG polypeptide variant may, if desired,comprise other modifications that are not related to such alterations.These may, for example, include truncation of the C-terminus by one ormore amino acid residues, addition of one or more extra residues at theN- and/or C-terminus, e.g. addition of a Met residue at the N-terminus,addition of the amino acid sequence Cys-Tyr-Cys at the N-terminus, aswell as “conservative amino acid substitutions”, i.e. substitutionsperformed within groups of amino acids with similar characteristics,e.g. small amino acids, acidic amino acids, polar amino acids, basicamino acids, hydrophobic amino acids and aromatic amino acids. Examplesof conservative substitutions in the present invention may in particularbe selected from the groups listed in the table below. 1 Alanine (A)Glycine (G) Serine (S) Threonine (T) 2 Aspartic acid (D) Glutamic acid(E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R) Histidine (H) Lysine(K) 5 Isoleucine (I) Leucine (L) Methionine Valine (V) (M) 6Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

The term “at least one” as used about a non-polypeptide moiety, an aminoacid residue, a substitution, etc is intended to mean one or more.

The term “AUC_(sc)” or “Area Under the Curve when administeredsubcutaneously” is used in its normal meaning, i.e. as the area underthe IFNG activity in serum-time curve, where the IFNG polypeptidevariant has been administered subcutaneously, in particular whenadministered subcutaneously in rats. Once the experimental IFNGactivity-time points have been determined, the AUC_(sc) may convenientlybe calculated by a computer program, such as GraphPad Prism 3.01.

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideis still present in the body/target organ, or the time at which theactivity of the polypeptide is 50% of the initial value.

As an alternative to determining functional in vivo half-life, “serumhalf-life” may be determined, i.e. the time at which 50% of thepolypeptide circulates in the plasma or bloodstream prior to beingcleared. Determination of serum half-life is often more simple thandetermining the functional in vivo half-life and the magnitude of serumhalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternatively terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”. The serum half-life mayconveniently by determined in rats, cf. the Materials and Method sectionherein. It is important to note that the term “serum half-life”, whenused herein, for a given IFNG polypeptide variant must be determined fora sample that has been administered intravenously (iv).

The term “serum” is used in its normal meaning, i.e. as blood plasmawithout fibrinogen and other clotting factors.

The polypeptide is normally cleared by the action of one or more of thereticuloendothelial systems (RES), kidney, spleen or liver, or byspecific or unspecific proteolysis. The term “renal clearance” is usedin its normal meaning to indicate any clearance taking place by thekidneys, e.g. by glomerular filtration, tubular excretion or tubularelimination. Normally, renal clearance depends on physicalcharacteristics of the polypeptide, including molecular weight, size(relative to the cutoff for glomerular filtration), symmetry,shape/rigidity, charge, attached carbohydrate chains and the presence ofcellular receptors for the polypeptide. A molecular weight of about 67kDa is normally considered to be a cut-off-value for renal clearance.Renal clearance may be measured by any suitable assay, e.g. anestablished in vivo assay. For instance, renal clearance may bedetermined by administering a labelled (e.g. radiolabelled orfluorescence labelled) conjugated polypeptide to a patient and measuringthe label activity in urine collected from the patient. Reduced renalclearance is determined relative to the reference molecule, such ashuIFNG, [S99T]huIFNG or Actimmune®. The functionality to be retained isnormally selected from antiviral, antiproliferative, immunomodulatory orIFNG receptor binding activity.

The term “increased” as used about the functional in vivo half-life orserum half-life is used to indicate that the relevant half-life of theIFNG variant is statistically significantly increased relative to thatof a reference molecule, such as glycosylated huIFNG (SEQ ID NO:17),glycosylated [S99T]huIFNG (SEQ ID NO:1) or Actimmune® (SEQ IDNO:34-produced in E. coli), when administered intravenously and whendetermined under comparable conditions. Thus, interesting IFNGpolypeptide variants are such variants, which, has an increasedfunctional in vivo half-life or an increased serum half-life as comparedto any of the reference molecules mentioned above.

More particularly, interesting IFNG variants are such variants where theratio between the serum half-life (or functional in vivo half-life) ofsaid variant and the serum half-life (or functional in vivo half-life)of huIFNG or [S99T]huIFNG in their glycosylated forms is at least 1.25,more preferably at least 1.50, such as at least 1.75, e.g. at least 2,even more preferably at least 3, such as at least 4, e.g. at least 5,when administered intravenously, in particular when administeredintravenously in rats.

Other examples of interesting IFNG variants are such variants where theratio between the serum half-life (or functional in vivo half-life) ofsaid variant and the serum half-life (or functional in vivo half-life)of Actimmune® (SEQ ID NO:34—produced in E. coli) is at least 2 morepreferably at least 3, such as at least 4, e.g. at least 5, even morepreferably at least 6, such as at least 7, e.g. at least 8, mostpreferably at least 9, such as at least 10, when administeredintravenously, in particular when administered intravenously in rats.

The term “increased” as used about the AUC_(sc) is used to indicate thatthe Area Under the Curve for an IFNG variant of the invention, whenadministered subcutaneously, is statistically significantly increasedrelative to that of a reference molecule, such as glycosylated huIFNG(SEQ ID NO:17), glycosylated [S99T]huIFNG (SEQ ID NO:1) or Actimmune®(SEQ ID NO:34—produced in E. coli), determined under comparableconditions. Thus, preferred IFNG variants are such variants, which havean increased AUC_(sc), as compared to any of the reference moleculesmentioned above. Evidently, the same amount of IFNG activity should beadministered for the IFNG variant of the invention and the referencemolecule. Consequently, in order to make direct comparisons betweendifferent IFNG molecules, the AUC_(sc) values may be normalized, i.e.they may be expressed as AUC_(sc)/dose administered.

Particular preferred IFNG variants are such variants where the ratiobetween the AUC_(sc) of said variant and the AUC_(sc) of glycosylatedhuIFNG or glycosylated [S99T]huIFNG is at least 1.25, such as at least1.5, e.g. at least 2, more preferably at least 3, such as at least 4,e.g. at least 5 or at least 6, even more preferably at least 7, such asat least 8, e.g. at least 9 or at least 10, most preferably at least 12,such as at least 14, e.g. at least 16, at least 18 or at least 20, inparticular when administered (subcutaneously) in rats.

Other examples of particular preferred IFNG variants are such variantswhere the ratio between the AUC_(sc) of said variant and the AUC_(sc) ofActimmune® is at least 100, more preferably at least 150, such as atleast 200, e.g. at least 250, even more preferably at least 300, such asat least 400 e.g. at least 500, most preferably at least 750, such as atleast 1000, e.g. at least 1500 or at least 2000, in particular whenadministered (subcutaneously) in rats.

The term “T_(max,sc)” is used about the time in the IFNG activity inserum-time curve when the highest IFNG activity in serum is observed.Preferred IFNG variants of the invention, are such variants which havean increased T_(max,sc) as compared to Actimmune® and/or as compared toglycosylated huIFNG. More particularly, such preferred variants have aT_(max,sc) (when determined after subcutaneous administration in rats)of at least 200 min, such as at least 250 min, e.g. at least 300 min,more preferably at least 350 min, such as at least 400 min.

The term “reduced immunogenicity” is intended to indicate that the IFNGpolypeptide variant gives rise to a measurably lower immune responsethan a reference molecule, e.g. huIFNG or Actimmune®, as determinedunder comparable conditions. The immune response may be a cell orantibody mediated response (see, e.g., Roitt: Essential Immunology (8thEdition, Blackwell) for further definition of immunogenicity). Normally,reduced antibody reactivity is an indication of reduced immunogenicity.Reduced immunogenicity may be determined by use of any suitable methodknown in the art, e.g. in vivo or in vitro.

In the present context the terms “increased glycosylation”, “increaseddegree of in vivo N-glycosylation” or “increased degree ofN-glycosylation” are intended to indicate increased levels of attachedcarbohydrate molecules, normally obtained as a consequence of increased(or better) utilization of glycosylation site(s). It is well-known(Hooker et al., 1998, J. Interferon and Cytokine Res. 18, 287-295 andSarenva et al., 1995, Biochem J., 308, 9-14) that when huIFNG isexpressed in CHO cells only about 50% of the IFNG molecules utilizesboth glycosylation sites, about 40% utilizes one glycosylation site(1N), and about 10% is not glycosylated (0N). The increased degree of invivo N-glycosylation may be determined by any suitable method known inthe art, e.g. by SDS-PAGE. One convenient assay for determiningincreased glycosylation is the method described in the section entitled“Determination of Increased Glycosylation” in the Materials and Methodssection herein.

When used herein the term “population of IFNG polypeptide variants” or“composition comprising a population of IFNG polypeptide variants” isintended to cover a composition comprising at least two IFNGpolypeptides glycosylated to a different extent. As will be understoodthe present invention provides means for obtaining a population of IFNGpolypeptides, wherein an increased amount of the INFG molecules presentin the population is fully glycosylated.

Thus, the present invention also relates to a homogeneous population ofIFNG polypeptides of the invention (i.e. a population, wherein most ofthe IFNG polypeptides are fully glycosylated) or a compositioncomprising a homogenous population of IFNG polypeptides of theinvention. For example, the population of IFNG polypeptides may containat least 70% of the IFNG polypeptide of the invention, preferably atleast 75%, such as at least 80%, e.g. at least 85%, more preferably atleast 90%, such as at least 95%, e.g. at least 96%, even more preferablyat least 97%, such as at least 98%, e.g. at least 99%.

The term “exhibiting IFNG activity” is intended to indicate that thepolypeptide variant has one or more of the functions of native huIFNG orrhuIFNG, including the capability to bind to an IFNG receptor and causetransduction of the signal transduced upon huIFNG-binding of itsreceptor as determined in vitro or in vivo (i.e. in vitro or in vivobioactivity). The IFNG receptor has been described by Aguet et al. (Cell55:273-280, 1988) and Calderon et al. (Proc. Natl. Acad. Sci. USA85:4837-4841, 1988). A suitable assay for testing IFNG activity is theassay entitled “Primary Assay” disclosed herein. When using the “PrimaryAssay” described herein, polypeptide variants “exhibiting IFNG activity”have a specific activity of at least 5% as compared to the rhuIFNG. Itwill be understood, that depending on which specific modifications areperformed, for example whether the variant is PEGylated or not, may leadto activities over a wide range. Thus, examples of specific activitiesmay range from as low as 5% to as high as 150% as compared to rhuIFNG.For example, the specific activity may be at least 10% (e.g. 10-125%),such as at least 15% (e.g. 15-125%), e.g. at least 20% (such as20-125%), at least 25% (e.g. 25-125%), at least 30% (e.g. 30-125%), atleast 35% (e.g. 35-125%), at least 40% (e.g. 40-125%), at least 45%(e.g. 45-125%), at least 50% (e.g. 50-125%), at least 55% (e.g.55-125%), at least 60% (e.g. 60-125%), at least 65% (e.g. 65-125%), atleast 70% (e.g. 70-125%), at least 75% (e.g. 75-125%), at least 80%(e.g. 80-125%) or at least 90% (e.g. 90-110%) as compared to thespecific activity of rhuIFNG.

An “IFNG polypeptide” is a polypeptide exhibiting IFNG activity, i.e.the term “IFNG polypeptide” is used about any IFNG molecule(independently of whether this molecule is huIFNG, a truncated formthereof, or a variant thereof) as long as said IFNG molecule exhibitsIFNG activity as defined herein. The term “IFNG polypeptide” is usedherein about the polypeptide in monomer or dimeric form, as appropriate.For instance, when specific substitutions are indicated these arenormally indicated relative to the huIFNG polypeptide monomer. Whenreference is made to the IFNG molecule of the invention this is normallyin dimeric form (and thus, e.g., comprises two IFNG polypeptide monomersmodified as described). The dimeric form of the IFNG polypeptides may beprovided by the normal association of two monomers or be in the form ofa single chain dimeric IFNG polypeptide.

The term “parent” is intended to indicate the IFNG polypeptide to havethe glycosylation site(s) improved in accordance with the presentinvention. Although the parent polypeptide to be modified by the presentinvention may be any polypeptide with IFNG activity, and thus be derivedfrom any origin, e.g. a non-human mammalian origin, it is preferred thatthe parent polypeptide is huIFNG with the amino acid sequence shown inSEQ ID NO:17 or a fragment thereof.

A “fragment” is a part of the full-length IFNG polypeptide sequence(e.g. a fragment of the full-length huIFNG polypeptide shown in SEQ IDNO:17 or a fragment of the full-length [S99T]huIFNG polypeptide variantshown in SEQ ID NO:1) exhibiting IFNG activity, e.g. a C-terminally orN-terminally truncated version thereof. Specific examples of IFNGpolypeptide variant fragments include [S99T]huIFNG C-terminallytruncated with 1-15 amino acid residues, e.g. with 1 amino acid residue(SEQ ID NO:2), 2 amino acid residues (SEQ ID NO:3), 3 amino acidresidues (SEQ ID NO:4), 4 amino acid residues (SEQ ID NO:5), 5 aminoacid residues (SEQ ID NO:6), 6 amino acid residues (SEQ ID NO:7), 7amino acid residues (SEQ ID NO:8), 8 amino acid residues (SEQ ID NO:9),9 amino acid residues (SEQ ID NO:10), 10 amino acid residues (SEQ IDNO:11), 11 amino acid residues (SEQ ID NO:12), 12 amino acid residues(SEQ ID NO:13), 13 amino acid residues (SEQ ID NO:14), 14 amino acidresidues (SEQ ID NO:15) or 15 amino acid residues (SEQ ID NO:16) and/orN-terminally truncated with 1-3 amino acid residues. Specific examplesof huIFNG fragments include huIFNG, which is C-terminally truncated with1-15 amino acid residues, e.g. with 1 amino acid residue (SEQ ID NO:19),2 amino acid residues (SEQ ID NO:10), 3 amino acid residues (SEQ IDNO:21), 4 amino acid residues (SEQ ID NO:22), 5 amino acid residues (SEQID NO:23), 6 amino acid residues (SEQ ID NO:24), 7 amino acid residues(SEQ ID NO:25), 8 amino acid residues (SEQ ID NO:26), 9 amino acidresidues (SEQ ID NO:27), 10 amino acid residues (SEQ ID NO:28), 11 aminoacid residues (SEQ ID NO:29), 12 amino acid residues (SEQ ID NO:30), 13amino acid residues (SEQ ID NO:31), 14 amino acid residues (SEQ IDNO:32) or 15 amino acid residues (SEQ ID NO:33) and/or N-terminallytruncated with 1-3 amino acid residues.

As indicated above, the IFNG polypeptide variant may comprise at leastone further modification in addition to the S99T substitution as long assaid variant exhibits IFNG activity, i.e. the variant may be a variantof [S99T]huIFNG, or a variant of a fragment of [S99T]huIFNG. Specificexamples of such variants are variants having introduced and/or removedamino acid residues comprising an attachment group for a non-polypeptidemoiety. Other examples of variants of [S99T]huIFNG (and fragmentsthereof) are the variants described in the “Background of the invention”section above and include, e.g. [S99T]huIFNG with the N-terminaladdition Cys-Tyr-Cys or with Met, and the cysteine-modified variantsdisclosed in U.S. Pat. No. 6,046,034.

Normally, the variant according to the invention is encoded by anucleotide sequence, which, compared to the nucleotide sequence encodingthe parent IFNG polypeptide, has been modified in accordance with thepresent invention.

This may, however, not always be the case, since the variant polypeptidemay be subjected to C- or N-terminal truncation during posttranslationalprocessing, e.g. by C- or N-terminal cleavage by proteases in the cell,in the expression media, during purification, etc., so that theresulting variant polypeptide is a truncated version of the originallyproduced variant polypeptide (for example, although a full-lengthvariant is initially produced, a C-terminally truncated variantpolypeptide may be obtained due to posttranslational processing of thefull-length variant polypeptide). In this case, the term “parent” shouldbe construed as the truncated form to be modified in accordance with theinvention.

The term “variant” is intended to cover a polypeptide, which differs inone or more amino acid residues from its parent polypeptide (normallySEQ ID NO:17 or any of the truncated forms thereof shown in SEQ IDNOS:19-33), typically in 1-15 amino acid residues (such as in 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues), e.g. in1-10 amino acid residues, in 1-5 amino acid residues or in 1-3 aminoacid residues.

The term “functional site” is intended to indicate one or more aminoacid residues which is/are essential for, or otherwise involved in, thefunction or performance of IFNG. Such amino acid residues are “locatedat” the functional site. The functional site may be determined bymethods known in the art and is preferably identified by analysis of astructure of the polypeptide complexed to a relevant receptor, such asthe IFNG receptor.

The term “huIFNG” is intended to mean the mature form of wild-type humanIFNG having the amino sequence shown in SEQ ID NO:17.

The term “rhuIFNG” is intended to cover the mature form of wild-typehuman IFNG having the amino acid sequence shown in SEQ ID NO:17, whichhas been produced by recombinant means.

The term “[S99T]huIFNG” is used to indicate the mature form of wild-typehuman IFNG, wherein the serine residue in position 99 has been replacedwith a threonine residue (disclosed in SEQ ID NO:1).

When used herein the term “glycoyslated huIFNG” indicates that thehuIFNG polypeptide is produced in a cell capable of glycosylating thepolypeptide and, therefore, the huIFNG polypeptide is glycosylated atits native N-glycosylation sites (position 25 and 97 of SEQ ID NO:17).

In a similar way, the term “glycoyslated huIFNG variant” indicates thatthe IFNG polypeptide variant is produced in a cell capable ofglycosylating the polypeptide variant.

When used herein the term “Actimmune®” refers to the 140 amino acid form(Actimmune® is C-terminally truncated with 4 amino acid residues andincludes one N-terminal Met residue) of IFNG (disclosed in SEQ ID NO:34)achieved by fermentation of a genetically engineered E. coli bacterium.Further information of Actimmune® is available on www.actimmune.com.

Interferon Gamma Polypeptide Variants of the Present Invention

IFNG Variants of the Invention with Optimised In Vivo GlycosylationSites

As indicated previously, it has surprisingly been found thatglycosylation of the naturally occurring N-glycosylation site located inposition 97 of huIFNG may be increased, i.e. an increased fraction offully, or substantially fully, glycosylated IFNG molecules may beobtained, by substituting the serine residue located in position 99 ofhuIFNG (or fragments thereof) with a threonine residue. Inspection ofFIG. 1 reveals that a significant increase in the degree of fullyglycosylated IFNG polypeptide can be achieved. For the [S99T]huIFNG (SEQID NO:1) polypeptide variant it can be seen that about 90% of thepolypeptide variants present in the harvested medium utilized bothN-glycosylation site, whereas only about 60% of the rhuIFNG polypeptidespresent in the harvested medium was fully glycosylated.

Accordingly, in a first aspect the present invention relates to an IFNGpolypeptide variant exhibiting IFNG activity and having the amino acidsequence shown in SEQ ID NO:1 (i.e. [S99T]huIFNG), or fragment thereofexhibiting IFNG activity.

As already discussed above, it is known that C-terminally truncatedforms of huIFNG retain activity, and in some cases even have increasedactivity, compared to huIFNG. Thus, in an interesting embodiment of theinvention the IFNG polypeptide variant of the invention is a fragment ofSEQ ID NO:1, which is C-terminally truncated with 1-15 amino acidresidues, typically C-terminally truncated with 1-10 amino acidresidues. Specific examples of such C-terminally truncated forms of SEQID NO:1 are disclosed in SEQ ID NOS:2-16. The IFNG polypeptide fragmentaccording to this embodiment of the present invention exhibits IFNGactivity.

It will be understood that the glycosylated IFNG polypeptide variantsaccording to this aspect should be expressed recombinantly in aglycosylating host cell, preferably a mammalian host cell, such as anyof those mentioned in the section entitled “Coupling to a sugar moiety”.

As explained above, only about 50-60% of the total population ofexpressed IFNG polypeptides are fully glycosylated when rhuIFNG isexpressed in CHO cells. Thus, one of the main advantages of the IFNGpolypeptide variants of the present invention is the higher utilizationof the position 97 in vivo N-glycosylation site, which in turn has theconsequence that a more homogenous population is obtained compared tohuIFNG. Due to the more homogenous population, compositions (e.g. theharvested medium) comprising such a population of IFNG polypeptidevariants do not require the same cumbersome and time-consumingpurification as rhuIFNG.

Thus, in a further aspect the present invention relates to a populationof IFNG polypeptide variants, or to a composition comprising apopulation of IFNG polypeptide variants, wherein said populationcomprises at least about 70% of an IFNG polypeptide variant of theinvention. Preferably, the composition comprises at least 75%, morepreferably at least 80%, even more preferably at least 85%, such asabout 90% of an IFNG polypeptide variant of the invention.

More particularly, the invention relates to a population of IFNGpolypeptide variants, or to a composition comprising a population ofIFNG polypeptide variants, wherein said population comprises at least70%, preferably at least 75%, more preferably at least 80%, even morepreferably at least 85%, such as about 90% of the IFNG polypeptidevariant having the amino acid sequence shown in SEQ ID NO:1.

Analogously, the invention also relates to a population of IFNGpolypeptide variants, or to a composition comprising a population ofIFNG polypeptide variants, wherein said population comprises at least70%, preferably at least 75%, more preferably at least 80%, even morepreferably at least 85%, such as about 90% of an IFNG polypeptidevariant fragment having an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.

In addition to the already mentioned S99T mutation required foroptimisation of the in vivo N-glycosylation site at position 97 inrhuIFNG, other in vivo glycosylation sites, which have been introducedinto SEQ ID NO:1 or fragments thereof (e.g. in order to increase theserum half-life and/or to increase the AUC_(sc)) may be optimised.Normally, the in vivo glycosylation site is an N-glycosylation site, butalso an O-glycosylation site is contemplated as relevant for the presentinvention. This optimisation may be achieved by performing amodification, preferably a substitution, in a position, which is locatedclose to a glycosylation site, in particular close to an in vivoN-glycosylation. Typically, such an in vivo N-glycoyslation site is anintroduced in vivo N-glycosylation site. Specific examples of suitablepositions to introduce in vivo N-glycosylation sites are disclosed in WO01/36001 and further below.

An amino acid residue “located close to” a glycosylation site is usuallylocated in position −4, −3, −2, −1, +1, +2, +3 or +4 relative to theamino acid residue of the glycosylation site to which the carbohydrateis attached, preferably in position −1, +1, or +3, in particular inposition +1 or +3. Thus, the amino acid residue located close to an invivo N-glycosylation site (having the sequence N-X-S/T/C) may be locatedin position −4, −3, −2, −1, +1, +2, +3 or +4 relative to the N-residue.

When position +2 relative to the N-residue is modified it will beunderstood that only a limited number of modifications are possiblesince in order to maintain/introduce an in vivo N-glycosylation site,the amino acid residue in said position must be either Ser, Thr or Cys.

In a particular preferred embodiment of the invention, the modificationof the amino acid residue in position +2 relative to the in vivoN-glycosylation site is a substitution where the amino acid residue inquestion is replaced with a Thr residue. If, on the other hand, saidamino acid residue is already a Thr residue it is normally not preferredor necessary to perform any substitutions in that position. When X ismodified, X should not be Pro and preferably not Trp, Asp, Glu and Leu.Further, the amino acid residue to be introduced is preferably selectedform the group consisting of Phe, Asn, Gln, Tyr, Val, Ala, Met, Ile,Lys, Gly, Arg, Thr, His, Cys and Ser, more preferably Ala, Met, Ile,Lys, Gly, Arg, Thr, His, Cys and Ser, in particular Ala or Ser.

When position +3 relative to the N-residue is modified, the amino acidresidue to be introduced is preferably selected from the groupconsisting of His, Asp, Ala, Met, Asn, Thr, Arg, Ser and Cys, morepreferably Thr, Arg, Ser and Cys. Such modifications are particularrelevant if the X residue is a Ser residue.

Thus, with respect to the naturally present in vivo N-glycosylation, itis contemplated that the N-glycosylation site at position 97 may befurther optimised by performing a modification, such as a substitution,in a position selected from the group consisting of E93, K94, L95, T96,Y98, V100 and T101 (i.e. at position −4, −3, −2, −1, +1, +3 or +4relative to N97). Specific examples of substitutions performed inposition 98 of SEQ ID NO:1 (or fragments thereof) include Y98F, Y98N,Y98Q, Y98V, Y98A, Y98M, Y981, Y98K, Y98G, Y98R, Y98T, Y98H, Y98C andY98S, preferably Y98A, Y98M, Y981, Y98K, Y98G, Y98R, Y98T, Y98H, Y98Cand Y98S, in particular Y98S. Specific examples of substitutionsperformed in position 100 of SEQ ID NO:1 (or fragments thereof) includeV100H, V100D, V100A, V100M, V100N, V100T, V100R, V100S, or V100C, inparticular V100T, V100R, V100S or V100C.

In a similar way, with respect to the in vivo N-glycosylation site atposition 25 it is contemplated that this site may be further optimisedby performing a modification, such as a substitution, in a positionselected from the group consisting of D21, V22, A23, D24, G26, L28 andF29 (i.e. at position −4, −3, −2, −1, +1, +3 or +4 relative to N25).Specific examples of substitutions performed in position 26 of SEQ IDNO:1 (or fragments thereof) include G26F, G26N, G26Y, G26Q, G26V, G26A,G26M, G26I, G26K, G26R, G26T, G26H, G26C and G26S, preferably G26A,G26M, G26I, G26K, G26R, G26T, G26H, G26C and G26S, more preferably G26Aand G26S, in particular G26A. Specific examples of substitutionsperformed in position 28 of SEQ ID NO:1 (or fragments thereof) includeG28H, G28D, G28A, G28M, G28N, G28T, G28R, G28S, or G28S, in particularG28A, G28T, G28R, G28S or G28C.

Obviously, any of the modifications mentioned in connection withoptimisation of glycosylation at position 97 may be combined with any ofthe above-mentioned modifications performed in connection withoptimisation of glycosylation at position 25.

IFNG Variants of the Invention with Increased AUC_(sc) and/or IncreasedSerum Half-Life

In a further aspect the present invention relates to a variant of theIFNG polypeptide having the amino acid sequence shown in SEQ ID NO:1,wherein said variant exhibits IFNG activity.

Moreover, the present invention also relates to a variant of an IFNGpolypeptide fragment having an amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IDNO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ IDNO:16, wherein said variant exhibits IFNG activity.

Thus, such a variant comprises at least one further modificationcompared to SEQ ID NOS:1-16.

In order to avoid too much disruption of the structure and function ofthe [S99T]huIFNG polypeptide variant (or fragments thereof) the totalnumber of amino acid residues to be modified in accordance with thepresent invention typically does not exceed 15. Usually, the IFNGpolypeptide variant comprises 1-10 modifications relative to the aminoacid sequence shown in SEQ ID NO:1, such as 1-8, 2-8, 1-5, 1-3 or 2-5modifications relative to the amino acid sequence shown in SEQ ID NO:1.Preferably, the modification(s) is/are a substitution(s). It will beunderstood that similar considerations hold true for variants offragments of the IFNG polypeptide variant having the amino acid sequenceshown in SEQ ID NO:1. Thus, when the variant is a variant of any of thesequences disclosed in SEQ ID NOS:2-16, such a variant usually comprisesless than 15 modifications, typically 1-10 modifications, relative tothe relevant amino acid sequence shown in SEQ ID NOS:2-16, such as 1-8,2-8, 1-5, 1-3 or 2-5 modifications relative to the relevant the aminoacid sequence shown in SEQ ID NOS:2-16. Preferably, the modification(s)is/are a substitution(s).

Thus, normally such an IFNG polypeptide variant (i.e. a variant whichcomprises at least one further modification in addition to the S99Tsubstitution) comprises an amino acid sequence which differs from theamino acid sequence shown in SEQ ID NO:1 (or fragments thereof) in 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues.

In a preferred embodiment of the invention, the IFNG variant (further)comprises at least one introduced and/or at least one removed amino acidresidue comprising an attachment group for a non-polypeptide moiety.

By removing or introducing an amino acid residue comprising anattachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide so as to make the molecule moresusceptible to conjugation to the non-polypeptide moiety of choice, tooptimse the conjugation pattern (e.g. to ensure an optimal distributionof non-polypeptide moieties on the surface of the IFNG polypeptidevariant) and thereby obtain a new conjugate molecule, which exhibitsIFNG activity and in addition one or more improved properties ascompared to huIFNG- or rhuIFNG-based molecules available today. Forinstance, by introduction of attachment groups, the IFNG polypeptidevariant is boosted or otherwise altered in the content of the specificamino acid residues to which the relevant non-polypeptide moiety binds,whereby a more efficient, specific and/or extensive conjugation isachieved. By removal of one or more attachment groups it is possible toavoid conjugation to the non-polypeptide moiety in parts of thepolypeptide in which such conjugation is disadvantageous, e.g. to anamino acid residue located at or near a functional site of thepolypeptide (since conjugation at such a site may result in inactivationor reduced IFNG activity of the resulting conjugated polypeptide due toimpaired receptor recognition). Further, it may be advantageous toremove an attachment group located closely to another attachment groupin order to avoid heterogeneous conjugation to such groups. Ininteresting embodiments more than one amino acid residue of the IFNGpolypeptide is altered, e.g. the alteration embraces removal as well asintroduction of amino acid residues comprising attachment sites for thenon-polypeptide moiety of choice. This embodiment is considered ofparticular interest in that it is possible to specifically design theIFNG polypeptide variant so as to obtain an optimal conjugation to thenon-polypeptide moiety.

In addition to the removal and/or introduction of amino acid residuesthe polypeptide variant may comprise other modifications, e.g.substitutions, that are not related to introduction and/or removal ofamino acid residues comprising an attachment group for thenon-polypeptide moiety. Examples of such modifications includeconservative amino acid substitutions and/or introduction of Cys-Tyr-Cysor Met at the N-terminus.

The exact number of attachment groups available for conjugation andpresent in the IFNG polypeptide variant in dimeric form is dependent onthe effect desired to be achieved by the conjugation. The effect to beobtained is, e.g. dependent on the nature and degree of conjugation(e.g. the identity of the non-polypeptide moiety, the number ofnon-polypeptide moieties desirable or possible to conjugate to thepolypeptide, where they should be conjugated or where conjugation shouldbe avoided, etc.).

It will be understood that the amino acid residue comprising anattachment group for a non-polypeptide moiety, either it be removed orintroduced, is selected on the basis of the nature of thenon-polypeptide moiety part of choice and, in most instances, on thebasis of the conjugation method to be used. For instance, when thenon-polypeptide moiety is a polymer molecule such as a polyethyleneglycol- or polyalkylene oxide-derived molecule amino acid residuescapable of functioning as an attachment group may be selected from thegroup consisting of cysteine, lysine, aspartic acid, glutamic acid andarginine. In particular, cysteine is preferred. When the non-polypeptidemoiety is a sugar moiety the attachment group is, e.g. an in vivoglycosylation site, preferably an N-glycosylation site.

Whenever an attachment group for a non-polypeptide moiety is to beintroduced into or removed from the IFNG polypeptide having the aminoacid sequence shown as SEQ ID NO:1 (or fragments thereof), the positionof the polypeptide to be modified is conveniently selected as follows:

The position is preferably located at the surface of the IFNGpolypeptide, and more preferably occupied by an amino acid residue thathas more than 25% of its side chain exposed to the solvent, preferablymore than 50% of its side chain exposed to the solvent, as determined onthe basis of a 3D structure or model of IFNG in its dimeric form, thestructure or model optionally further comprising one or two IFNGreceptor molecules. Such positions are listed in Example 1 herein.

Also of interest is to modify any of the 23 C-terminal amino acidresidues of the parent IFNG polypeptide (in particular by introductionof amino acid residues comprising an attachment group for thenon-polypeptide moiety, such as Cys residues) since such residues arebelieved to be located at the surface of the IFNG polypeptide.

In addition, it may be of interest to modify one or more amino acidresidues located in the loop regions of the IFNG polypeptide since mostamino acid residues within these loop regions are exposed to the surfaceand located sufficiently far away from functional sites so thatnon-polypeptide moieties, such as polymer molecules, in particular PEGmolecules, and/or N-glycosylation sites, may be introduced withoutimpairing the function of the molecule. Such loops regions may beidentified by inspection of the three-dimensional structure of huIFNG.The amino acid residues constituting said loop regions are residuesN16-K37 (the “A-B loop”), F60-S65 (the “B-C loop”), N83-S84 (the “C-Dloop”) and Y98-L103 (the “D-E loop”).

The amino acid residues constituting the IFNG receptor binding site areQ1, D2, Y4, V5, E9, K12, G18, H19, S20, D21, V22, A23, D24, N25, G26,T27, L30, K34, K37, K108, H111, E112, I114, Q115, A118, E119 (see alsoExample 2 herein). In general, it is preferred that attachment groupsfor a non-polypeptide moiety (such as additional N-glycosylation sitesand/or cysteine residues) are not introduced into this site of themolecule.

In order to determine an optimal distribution of attachment groups, thedistance between amino acid residues located at the surface of the IFNGpolypeptide is calculated on the basis of a 3D structure of the IFNGdimeric polypeptide. More specifically, the distance between the CB's ofthe amino acid residues comprising such attachment groups, or thedistance between the functional group (NZ for lysine, CG for asparticacid, CD for glutamic acid, SG for cysteine) of one and the CB ofanother amino acid residue comprising an attachment group aredetermined. In case of glycine, CA is used instead of CB. In the IFNGpolypeptide part of the invention any of said distances is preferablymore than 8 Å, in particular more than 10 Å in order to avoid or reduceheterogeneous conjugation.

Also, the amino acid sequence of the IFNG polypeptide variant may differfrom that of SEQ ID NO:1 (or fragments thereof) in that one or moreamino acid residues constituting part of an epitope has been removed,preferably by substitution to an amino acid residue comprising anattachment group for the non-polypeptide moiety, so as to destroy orinactivate the epitope. Epitopes of [S99T]huIFNG, huIFNG or rhuIFNG maybe identified by use of methods known in the art, also known as epitopemapping, see, e.g. Romagnoli et al., Biol Chem, 1999, 380(5):553-9,DeLisser H M, Methods Mol Biol, 1999, 96:11-20, Van de Water et al.,Clin Immunol Immunopathol, 1997, 85(3):229-35, Saint-Remy J M,Toxicology, 1997, 119(1):77-81, and Lane D P and Stephen C W, Curr OpinImmunol, 1993, 5(2):268-71. One method is to establish a phage displaylibrary expressing random oligopeptides of e.g. 9 amino acid residues.IgG1 antibodies from specific antisera towards [S99T]huIFNG, huIFNG orrhuIFNG are purified by immunoprecipitation and the reactive phages areidentified by immunoblotting. By sequencing the DNA of the purifiedreactive phages, the sequence of the oligopeptide can be determinedfollowed by localization of the sequence on the 3D-structure of theIFNG. The thereby identified region on the structure constitutes anepitope that then can be selected as a target region for introduction ofan attachment group for the non-polypeptide moiety.

Functional in vivo half-life and serum half-life is e.g. dependent onthe molecular weight of the polypeptide variant and the number ofattachment groups needed for providing increased half-life may depend onthe molecular weight of the non-polypeptide moiety in question. In oneembodiment, the IFNG polypeptide variant of the invention has amolecular weight of at least 67 kDa, in particular at least 70 kDa asmeasured by SDS-PAGE according to Laemmli, U.K., Nature Vol 227 (1970),p 680-85. IFNG has a Mw in the range of about 34-50 kDa, and thereforeadditional about 20-40 kDa is required to obtain the desired effect.This may, e.g., be provided by 2-4 10 kDa PEG molecules or by acombination of additional in vivo glycosylation sites and additional PEGmolecules, or as otherwise described herein.

Preferably, a conjugated IFNG polypeptide variant according to theinvention comprises 1-10 (additional) non-polypeptide moieties, such as1-8, 2-8, 1-5, 1-3 or 2-5 (additional) non-polypeptide moieties.Typically, a conjugated variant comprises 1-3 (additional)non-polypeptide moieties, such as 1, 2 or 3 (additional) non-polypeptidemoieties.

As mentioned above, under physiological conditions huIFNG exists as adimeric polypeptide. The polypeptide is normally in homodimeric form(e.g. prepared by association of two IFNG polypeptide molecules preparedas described herein). However, if desired the IFNG polypeptide variantmay be provided in single chain form, wherein two IFNG polypeptidemonomers are linked via a peptide bond or a peptide linker. Providingthe IFNG polypeptide variant in single chain form has the advantage thatthe two constituent IFNG polypeptides may be different which can beadvantageous, e.g., to enable asymmetric mutagenesis of thepolypeptides. For instance, PEGylation sites can be removed from thereceptor-binding site from one of the monomers, but retained in theother. Thereby, after PEGylation one monomer has an intactreceptor-binding site, whereas the other may be fully PEGylated (andthus provide significantly increased molecular weight).

IFNG Variants of the Invention Wherein the Non-Polypeptide Moiety is aSugar Moiety

In a preferred embodiment of the invention the IFNG variant of SEQ IDNO:1 (or fragments thereof) comprises at least one introducedglycosylation site and/or at least one removed glycosylation site.Preferably, the glycosylation site is an in vivo N-glycosylation site,i.e. the non-polypeptide moiety is a sugar moiety, e.g. an O-linked orN-linked sugar moiety, preferably an N-linked sugar moiety.

In one interesting embodiment of the invention said variant comprises atleast one introduced glycosylation site, in particular an introduced invivo N-glycosylation site. Preferably, the introduced glycosylation siteis introduced by a substitution.

For instance, an in vivo N-glycosylation site may be introduced into aposition of the IFNG polypeptide of SEQ ID NO:1 (of fragments thereof)comprising an amino acid residue exposed to the surface. Preferably saidsurface-exposed amino acid residue has at least 25% of the side chainexposed to the surface, in particular at least 50% of its side chainexposed to the surface. Details regarding determination of suchpositions can be found in Example 1 herein.

The N-glycosylation site is introduced in such a way that the N-residueof said site is located in said position. Analogously, anO-glycosylation site is introduced so that the S or T residue making upsuch site is located in said position. It should be understood that whenthe term “at least 25% (or 50%) of its side chain exposed to thesurface” is used in connection with introduction of an in vivoN-glycosylation site this term refers to the surface accessibility ofthe amino acid side chain in the position where the sugar moiety isactually attached. In many cases it will be necessary to introduce aserine or a threonine residue in position +2 relative to the asparagineresidue to which the sugar moiety is actually attached and thesepositions, where the serine or threonine residues are introduced, areallowed to be buried, i.e. to have less than 25% (or 50%) of their sidechains exposed to the surface of the molecule.

Furthermore, in order to ensure efficient glycosylation it is preferredthat the in vivo glycosylation site, in particular the N residue of theN-glycosylation site or the S or T residue of the O-glycosylation site,is located within the 118 N-terminal amino acid residues of the IFNGpolypeptide, more preferably within the 97 N-terminal amino acidresidues. Still more preferably, the in vivo glycosylation site isintroduced into a position wherein only one mutation is required tocreate the site (i.e. where any other amino acid residues required forcreating a functional glycosylation site is already present in themolecule).

For instance, substitutions that lead to introduction of an additionalN-glycosylation site at positions exposed at the surface of the IFNGpolypeptide and occupied by amino acid residues having at least 25% ofthe side chain exposed to the surface (in a structure with receptormolecule) include:

Q1N+P3S/T, P3N+V5S/T, K6N+A8S/T, E9N+L11S/T, K12S/T, K13N+F15S/T,Y14N+N16S/T, G18S/T, G18N, G18N+S20T, H19N+D21S/T, D21N+A23S/T,G26N+L28S/T, G31N+L33S/T, K34N+W36S/T, K37S/T, K37N+E39S/T, E38N,E38N+S40T, E39N+D41S/T, S40N+R42S/T, K55N+F57S/T, K58N+F60S/T, K61S/T,K61N+D63S/T, D62N+Q64S/T, D63N, D63N+S65T, Q64N+I66S/T, S65N+Q67S/T,Q67N, Q67N+S69T, K68N+V70S/T, E71N+I73S/T, T72N+K74S/T, K74N+D76S/T,E75N+M77S/T, K80S/T, V79N+F81S/T, K80N+F82S/T, N85S/T, S84N+K86S/T,K87S/T, K86N+K88S/T, K87N+R89S/T, D90N+F92S/T, E93N+L95S/T, K94N,K94N+T96S, T101N+L103S/T, D102N+N104S/T, L103N+V105S/T, Q106S/T, E119N,E119N+S121T, P122N+A124S/T, A123N+K125S/T, A124N, A124N+T126S,K125N+G127S/T, T126N+K128S/T, G127N+R129S/T, K128N+K130S/T,R129N+R131S/T, K130N, K130N+S132T, R131N+Q133S/T, S132N+M134S/T,Q133N+L135S/T, M134N+F136S/T, L135N+R137S/T, F136N+G138S/T,R137N+R139S/T, G138N+R140S/T, R139N+A141S/T, R140N and R140N+S142T, thesubstitution being indicated relative to [S99T]huIFNG with the aminoacid sequence shown in SEQ ID NO 1 (or relative to the relevant fragmentthereof having the amino acid sequence shown in SEQ ID NOS:2-16). S/Tindicates a substitution to a serine or threonine residue, preferably athreonine residue.

Substitutions that lead to introduction of an additional N-glycosylationsite at positions exposed at the surface of the IFNG polypeptide havingat least 50% of the side chain exposed to the surface (in a structurewith receptor molecule) include:

P3N+V5S/T, K6N+A8S/T, K12S/T, K13N+F15S/T, G18S/T, D21N+A23S/T,G26N+L28S/T, G31N+L33S/T, K34N+W36S/T, K37N+E39S/T, E38N, E38N+S40S/T,E39N+D41S/T, K55N+F57S/T, K58N+F60S/T, K61S/T, D62N+Q64S/T, Q64N+I66S/T,S65N+Q67S/T, K68N+V70S/T, E71N+I73S/T, E75N+M77S/T, N85S/T, S84N+K86S/T,K86N+K88S/T, K87N+R89S/T, K94N, K94N+T96S, T101N+L103S/T, D102N+N104S/T,L103N+V105S/T, Q106S/T, P122N+A124S/T, A123N+K125S/T, A124N,A124N+T126S, K125N+G127S/T, T126N+K128S/T, G127N+R129S/T, K128N+K130S/T,R129N+R131S/T, K130N, K130N+S132T, R131N+Q133S/T, S132N+M134S/T,Q133N+L135S/T, M134N+F136S/T, L135N+R137S/T, F136N+G138S/T,R137N+R139S/T, G138N+R140S/T, R139N+A141S/T, R140N and R140N+S142T, thesubstitution being indicated relative to [S99T]huIFNG with the aminoacid sequence shown in SEQ ID NO 1 (or relative to the relevant fragmentthereof having the amino acid sequence shown in SEQ ID NOS:2-16). S/Tindicates a substitution to a serine or threonine residue, preferably athreonine residue.

Substitutions where only one amino acid substitution is required tointroduce an N-glycosylation site include K12S/T, G18S/T, G18N, K37S/T,E38N, M45N, I49N, K61S/T, D63N, Q67N, V70N, K80S/T, F82N, N85S/T,K87S/T, K94N, Q106S/T, E119N, A124N, K130N and R140N, in particularK12S/T, G18N, G18S/T, K37S/T, E38N, K61S/T, D63N, Q67N, K80S/T, N85S/T,K94N, Q106S/T, A124N, K130N, and R140N (positions with more than 25% ofits site chain exposed to the surface (in a structure without receptormolecule)), or more preferably G18N, E38N, D63N, Q67N, K94N, S99N,A124N, K130N and R140N (positions with more than 50% of its side chainexposed to the surface in a structure without receptor molecule).

Usually, it is not preferred to introduce N-glycosylation sites in theregion constituting the receptor binding site (except in special cases,cf. the section entitled “Variants with a reduced receptor affinity”).Accordingly, the mutations Q1N+P3S/T, E9N+L11S/T, G18N, G18N+S20T,H19N+D21S/T, D21N+A23S/T, G26N+L28S/T, K34N+W36S/T, K37N+E39S/T, E119Nand E119N+S121T should normally not be performed, unless a reducedreceptor affinity is desired.

Particular preferred variants of the present invention include a variantof SEQ. ID NO:1 (or fragments thereof having the amino acid sequenceshown in SEQ ID NOS:2-16), wherein said variant exhibits IFNG activityand which comprises at least one substitution selected from the groupconsisting of K12S, K12T, G18S, G18T, E38N, E38N+S40T, K61S, K61T, N85S,N85T, K94N, Q106S and Q106T, more preferably selected from the groupconsisting of K12T, G18T, E38N+S40T, K61T, N85T, K94N and Q106T, evenmore preferably selected from the group consisting of K12T, G18T,E38N+S40T, K61T and N85T, in particular E38N+S40T.

In another interesting embodiment of the invention, the variant of SEQID NO:1 (or fragments thereof having the amino acid sequence shown inSEQ ID NOS:2-16) comprises at least two introduced glycosylation sites,in particular at least two introduced N-glycosylation sites. The atleast two modifications, in particular substitutions, leading to theintroduction of the at least two introduced N-glycosylation sites maypreferably be selected from the group consisting of K12S, K12T, G18S,G18T, E38N, E38N+S40T, K61S, K61T, N85S, N85T, K94N, Q106S and Q106T,more preferably selected from the group consisting of K12T, G18T,E38N+S40T, K61T, N85T, K94N and Q106T, even more preferably selectedfrom the group consisting of K12T, G18T, E38N+S40T, K61T and N85T.Specific examples of such substitutions giving rise to a variantcomprising at least two additional N-glycosylation sites include:K12T+G18T, K12T+E38N+S40T, K12T+K61T, K12T+N85T, G18T+E38N+S40T,G18T+K61T, G18T+N85T, E38N+S40T+K61T, E38N+S40T+N85T and K61T+N85T.

From the above lists of substitutions, it is preferable to selectsubstitutions located within the 118 N-terminal amino acid residues, inparticular within the 97 N-terminal amino acid residues.

The IFNG polypeptide variant of the invention may contain a singleadditional in vivo glycosylation site per monomer (as compared to SEQ IDNO:1 or fragments thereof). However, in order to become of a sufficientsize to increase the serum half-life it is often desirable that thepolypeptide comprises more than one additional in vivo N-glycosylationsite, in particular 2-7 or 2-5 additional in vivo N-glycosylation sites,such as 2, 3, 4 or 5 in vivo N-glycosylation sites. Such in vivoN-glycosylation sites are preferably introduced by one or moresubstitutions described in any of the above lists.

Furthermore, it will be understood that any of the above-mentionedmodifications may be combined with any of the modifications disclosed inthe section entitled “IFNG variant of the invention with optimised invivo glycosylation sites”, in particular with the substitution G26A.

IFNG Variants of the Invention Wherein the Non-Polypeptide Moiety is aMolecule, which has Cysteine as an Attachment Group

In another preferred embodiment of the invention the IFNG variant of SEQID NO:1 (or fragments thereof) comprises at least one introducedcysteine residue. For instance, a cysteine residue may be introducedinto a position of the IFNG polypeptide of SEQ ID NO:1 (or fragmentsthereof) comprising an amino acid residue exposed to the surface.Preferably said surface-exposed amino acid residue has at least 25% ofthe side chain exposed to the surface, in particular at least 50% of itsside chain exposed to the surface. Details regarding determination ofsuch positions can be found in Example 1 herein.

For instance, substitutions that lead to introduction of a cysteineresidue at positions exposed at the surface of the IFNG polypeptide andoccupied by amino acid residue having at least 25% of the side chainexposed to the surface (in a structure with receptor molecule) include:Q1C, D2C, P3C, K6C, E9C, N10C, K13C, Y14C, N16C, G18C, H19C, D21C, N25C,G26C, G31C, K34C, N35C, K37C, E38C, E39C, S40C, K55C, K58C, N59C, K61C,D62C, D63C, Q64C, S65C, Q67C, K68C, E71C, T72C, K74C, E75C, N78C, V79C,K80C, N83C, S84C, N85C, K86C, K87C, D90C, E93C, K94C, T101C, D102C,L103C, N104C and E119C, the substitution being indicated relative to[S99T]huIFNG with the amino acid sequence shown in SEQ ID NO 1 (orrelative to the relevant fragment thereof having the amino acid sequenceshown in SEQ ID NOS:2-16).

Substitutions that lead to introduction of a cysteine residue atpositions exposed at the surface of the IFNG polypeptide and occupied byamino acid residue having at least 50% of the side chain exposed to thesurface (in a structure with receptor molecule) include: P3C, K6C, N10C,K13C, N16C, D21C, N25C, G26C, G31C, K34C, K37C, E38C, E39C, K55C, K58C,N59C, D62C, Q64C, S65C, K68C, E71C, E75C, N83C, S84C, K86C, K87C, K94C,T101C, D102C, L103C and N104C, the substitution being indicated relativeto [S99T]huIFNG with the amino acid sequence shown in SEQ ID NO 1 (orrelative to the relevant fragment thereof having the amino acid sequenceshown in SEQ ID NOS:2-16).

Usually, it is not preferred to introduce a cysteine residue (andsubsequently attaching the cysteine residue to a non-polypeptide moiety)in the region constituting the receptor binding site (except in specialcases, cf. the section entitled “Variants with a reduced receptoraffinity”). Accordingly, the mutations Q1C, E9C, G18C, H19C, D21C, G26C,K34C, K37C and E119C should normally not be performed, unless a reducedreceptor affinity is desired.

More preferably, said cysteine residue is introduced by a substitutionselected from the group consisting of N10C, N16C, E38C, N59C, N83C,K94C, N104C and A124C.

In another interesting embodiment of the invention, the variant of SEQID NO:1 (or fragments thereof having the amino acid sequence shown inSEQ ID NOS:2-16) comprises at least two introduced cysteine residues.The at least two modifications, in particular substitutions, leading tothe introduction of the at least two cysteine residues may preferably beselected from the group consisting of N10C, N16C, E38C, N59C, N83C,K94C, N104C and A124C. Specific examples of such substitutions givingrise to a variant comprising at least two introduced cysteine residuesinclude: N10C+N16C, N10C+E38C, N10C+N59C, N10C+N83C, N10C+K94C,N10C+N104C, N10C+A124C, N16C+E38C, N16C+N59C, N16C+N83C, N16C+K94C,N16C+N104C, N16C+A124C, E38C+N59C, E38C+N83C, E38C+K94C, E38C+N104C,E38N+A124C, N59C+N83C, N59C+K94C, N59C+N104C, N59C+A124C, N83C+K94C,N83C+K94C, N83C+N104C, N83C+A124C, K94C+N104C, K94C+A124C andN104C+A124C.

As will be understood the introduced cysteine residue(s) may preferablybe conjugated to a non-polypeptide moiety, such as PEG or morepreferably mPEG. The conjugation between the cysteine-containingpolypeptide variant and the polymer molecule may be achieved in anysuitable manner, e.g. as described in the section entitled “Conjugationto a polymer molecule”, e.g. in using a one step method or in thestepwise manner referred to in said section. The preferred method forPEGylating the IFNG polypeptide variant is to covalently attach PEG tocysteine residues using cysteine-reactive PEGs. A number of highlyspecific, cysteine-reactive PEGs with different groups (e.g.orthopyridyl-disulfide, maleimide and vinylsulfone) and different sizePEGs (2-20 kDa, such as 5 kDa, 10 kDa, 12 kDa or 15 kDa) arecommercially available, e.g. from Shearwater Polymers Inc., Huntsville,Ala., USA).

It will be understood that any of the above-mentioned modifications maybe combined with any of the modifications disclosed in the sectionentitled “IFNG variants of the invention with optimised in vivoglycosylation sites”, in particular with the substitution G26A.

IFNG Variants of the Invention Wherein the First Non-Polypeptide Moietyis a Sugar Moiety and the Second Non-Polypeptide Moiety is a Molecule,which has Cysteine as an Attachment Group

In a further preferred embodiment of the invention the IFNG variant ofSEQ ID NO:1 (or fragments thereof) comprises at least one introducedN-glycosylation site and at least one introduced cysteine residue. Suchvariants may be prepared by selecting the residues described in the twopreceding sections describing suitable positions for introducingN-glycosylation sites and cysteine residues, respectively. However, in apreferred embodiment of the invention said variant comprisessubstitutions selected from the group consisting of K12T+N16C,K12T+E38C, K12T+N59C, K12T+N83C, K12T+K94C, K12T+N104C, K12T+A124C,G18T+N10C, G18T+E38C, G18T+N59C, G18T+N83C, G18T+K94C, G18T+N104C,G18T+A124C, E38N+S40T+N10C, E38N+S40T+N16C, E38N+S40T+N59C,E38N+S40T+N83C, E38N+S40T+K94C, E38N+S40T+N104C, E38N+S40T+A124C,K61T+N16C, K61T+N16C, K61T+E38C, K61T+N83C, K61T+K94C, K61T+N104C,K61T+A124C, N85T+N10C, N85T+N16C, N85T+E38C, N85T+N59C, N85T+K94C,N85T+N104C, N85T+A124C, K94N+N10C, K94N+N16C, K94N+E38C, K94N+N59C,K94N+N83C, K94N+N104C, K94N+A124C, Q106T+N10C, Q106T+N16C, Q106T+E38C,Q106T+N59C, Q106T+N83C, Q106T+K94C and Q106T+A124C, more preferably fromthe group consisting of E38N+S40T+N10C, E38N+S40T+N16C, E38N+S40T+N59C,E38N+S40T+N83C, E38N+S40T+K94C and E38N+S40T+N104C.

As will be understood the introduced cysteine residue may preferably beconjugated to a non-polypeptide moiety, such as PEG or more preferablymPEG. The conjugation between the cysteine-containing polypeptidevariant and the polymer may be achieved in any suitable manner, e.g. asdescribed in the section entitled “Conjugation to a polymer molecule”,e.g. in using a one step method or in the stepwise manner referred to insaid section. A suitable polymer is VS-mPEG or OPSS-mPEG.

Furthermore, it will be understood that any of the above-mentionedmodifications may be combined with any of the modifications disclosed inthe section entitled “IFNG variant of the invention with optimised invivo glycosylation sites”, in particular with the substitution G26A.

IFNG Variants with a Reduced Receptor Affinity

One way to increase the serum half-life of an IFNG polypeptide would beto decrease the receptor-mediated internalisation and thereby decreasethe receptor-mediated clearance.

The receptor-mediated internalisation is dependent upon the affinity ofthe IFNG dimer for the IFNG receptor complex and, accordingly, an IFNGvariant with a decreased affinity to the IFNG receptor complex isexpected to be internalised, and hence cleared, to a lesser extent.

The affinity of the IFNG dimmer to its receptor complex may be decreasedby performing one or more modifications, in particular substitutions, inthe receptor binding site of the IFNG polypeptide. The amino acidresidues which constitute the receptor binding site is defined inExample 2 herein. One class of substitutions that may be performed isconservative amino acid substitutions. In another embodiment, themodification performed gives rise to the introduction of anN-glycosylation site.

Thus, in a further particular preferred embodiment of the invention theIFNG variant of SEQ ID NO:1 (or fragments thereof) comprises at leastone modification, such as a substitution, in the receptor binding site(as defined herein). More particularly, the IFNG polypeptide comprisesat least one modification, preferably a substitution, which creates anin vivo N-glycosylation site, in said receptor binding site. Forinstance, such substitutions may be selected from the group consistingof Q1N+P3S/T, D2N+Y4S/T, Y4N+K6S/T, V5N+E7S/T, E9N+L11S/T, K12N+Y14S/T,G18N, G18N+S20T, H19N+D21S/T, S20N+V22S/T, D21N+A23S/T, V22N+D24S/T,D24N+G26S/T, G26N+L28S/T, L30N+132S/T, K34N+W36S/T, K37N+E39S/T,K108N+I110S/T, H111N+L113S/T, E112N+I114S/T, I114N+V116S/T,Q115N+M117S/T, A118N+L120S/T, E119N and E119N+S121T, preferably from thegroup consisting of Q1N+P3S/T, D2N+Y4S/T, E9N+L11S/T, K12N+Y14S/T, G18N,G18N+S20T, H19N+D21S/T, S20N+V22S/T, D21N+A23S/T, K34N+W36S/T,K37N+E39S/T, H111N+L113S/T, Q115N+M117S/T, A118N+L120S/T, E119N andE119N+S121T (introduction of N-glycosylation sites in positionscomprising an amino acid residue having at least 25% of its side chainexposed to the surface), more preferably from the group consisting ofQ1N+P3S/T, D2N+Y4S/T, E9N+L11S/T, G18N, G18N+S20T, H19N+D21S/T,S20N+V22S/T, D21N+A23S/T, K34N+W36S/T, K37N+E39S/T, Q115N+M117S/T,A118N+L120S/T, E119N and E119N+S121T (introduction of N-glycosylationsites in positions comprising an amino acid residue having at least 50%of its side chain exposed to the surface), even more preferably from thegroup consisting of Q1N+P3T, D2N+Y4T, E9N+L11T, G18N+S20T, H19N+D21T,S20N+V22T, D21N+A23T, K34N+W36T, K37N+E39T, Q115N+M117T, A118N+L120T andE119N+S121T, most preferably from the group consisting of G18N+S20T,H19N+D21T, D21N+A23T and E119N+S121T, in particular D21N+A23T.

Such variants are contemplated to exhibit a reduced receptor affinity ascompared to huIFNG or Actimmune®. The receptor affinity may be measuredby any suitable assay and will be known to the person skilled in theart. One example of a suitable assay for determining the receptorbinding affinity is the BIAcore® assay described in Michiels et al. Int.J. Biochem. Cell Biol. 30:505-516 (1998). Using the above-identifiedassay, IFNG variants considered useful for the purposes described hereinare such IFNG variants, wherein the binding affinity (K_(d)) is 1-95% ofthe K_(d)-value of glycosylated [S99T]huIFNG or Actimmune®. For examplethe K_(d)-value of the IFNG polypeptide may be 1-75% or 1-50%, such as1-25%, e.g. 1-20% or even as low as 1-15%, 1-10% or 1-5% of theK_(d)-value of glycosylated [S99T]huIFNG or Actimmune®.

Typically, such IFNG variants having reduced receptor affinity willexhibit a reduced IFNG activity, e.g. when tested in the “Primary Assay”described herein. For example, the IFNG polypeptide variant may exhibit1-95% of the specific activity of Actimunne® or rhuIFNG, e.g. 1-75%,such as 1-50%, e.g. 1-20% or 1-10% of the specific activity ofActimunne® or rhuIFNG.

As mentioned above, such IFNG variants are contemplated to possess anincreased serum-half due to the reduced receptor-mediated clearance.Therefore, the IFNG polypeptide variants according to the aspect of theinvention are contemplated to fulfil the requirements with respect toincreased serum-half described previously herein in connection with thedefinition of increased serum half-life.

Evidently, any of the above-mentioned modifications giving rise to areduced receptor binding affinity may be combined with any of the othermodifications disclosed herein, in particular the modificationsmentioned in the sections entitled “IFNG variants of the invention withoptimised N-glycosylation sites”, “IFNG variants of the inventionwherein the non-polypeptide moiety is a sugar moiety”, “IFNG variants ofthe invention wherein the non-polypeptide moiety is a molecule, whichhas cysteine as an attachment group” and “IFNG variants of the inventionwherein the first non-polypeptide moiety is a sugar moiety and thesecond non-polypeptide moiety is a molecule, which has cysteine as anattachment group”, such as G26A, E38N+S40T and combinations thereof.

Conjugation Methods

The Non-Polypeptide Moiety

As indicated further above the non-polypeptide moiety is preferablyselected from the group consisting of a polymer molecule, a lipophiliccompound, a sugar moiety (e.g. by way of in vivo N-glycosylation) and anorganic derivatizing agent. All of these agents may confer desirableproperties to the IFNG polypeptide variant, in particular increasedAUC_(sc), increased serum half-life and/or reduced immunogenicity. Thepolypeptide variant is normally conjugated to only one type ofnon-polypeptide moiety, but may also be conjugated to two or moredifferent types of non-polypeptide moieties, e.g. to a polymer moleculeand a sugar moiety, to a lipophilic group and a sugar moiety, to anorganic derivating agent and a sugar moiety, to a lipophilic group and apolymer molecule, etc. When conjugated to two different types ofnon-polypeptide moieties these are preferably a sugar moiety and apolymer moiety. The conjugation to two or more different non-polypeptidemoieties may be done simultaneous or sequentially. In the followingsections “Conjugation to a lipophilic compound”, “Conjugation to apolymer molecule”, “Conjugation to a sugar moiety” and “Conjugation toan organic derivatizing agent” conjugation to specific types ofnon-polypeptide moieties is described.

Conjugation to a Lipophilic Compound

The polypeptide variant and the lipophilic compound may be conjugated toeach other, either directly or by use of a linker. The lipophiliccompound may be a natural compound such as a saturated or unsaturatedfatty acid, a fatty acid diketone, a terpene, a prostaglandin, avitamine, a carotenoide or steroide, or a synthetic compound such as acarbon acid, an alcohol, an amine and sulphonic acid with one or morealkyl-, aryl-, alkenyl- or other multiple unsaturated compounds. Theconjugation between the polypeptide variant and the lipophilic compound,optionally through a linker may be done according to methods known inthe art, e.g. as described by Bodanszky in Peptide Synthesis, JohnWiley, New York, 1976 and in WO 96/12505.

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the polypeptide variant may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror heteropolymer, typically with a molecular weight in the range of300-100,000 Da or 1000-50,000 Da, such as in the range of 2000-40,000 Daor 2000-30,000 Da, e.g. in the range of 2000-20,000 Da, 2000-10,000 or1000-5000 Da. More particularly, the polymer molecule, such as PEG, inparticular MPEG, will typically have a molecular weight of about 2, 5,10, 12, 15, 20, 30, 40 or 50 kDa, in particular a molecular weight ofabout 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa or 20 about kDa.

When used about polymer molecules herein, the word “about” indicates anapproximate average molecular weight and reflects the fact that therewill normally be a certain molecular weight distribution in a givenpolymer preparation.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer, which comprises one or more differentcoupling groups, such as, e.g., a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs, poly-vinyl alcohol (PVA),poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleic acidanhydride, polystyrene-co-malic acid anhydride, dextran includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Another example of a polymer molecule is human albuminor another abundant plasma protein. Generally, polyalkyleneglycol-derived polymers are biocompatible, non-toxic, non-antigenic,non-immunogenic, have various water solubility properties, and areeasily excreted from living organisms.

PEG is the preferred polymer molecule to be used, since it has only fewreactive groups capable of cross-linking compared, e.g., topolysaccharides such as dextran, and the like. In particular,monofunctional PEG, such as, monomethoxypolyethylene glycol (mPEG), isof interest since its coupling chemistry is relatively simple (only onereactive group is available for conjugating with attachment groups onthe polypeptide). Consequently, the risk of cross-linking is eliminated,the resulting conjugated polypeptide variantss are more homogeneous andthe reaction of the polymer molecules with the polypeptide is easier tocontrol.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide variant, the hydroxyl end groups of the polymer moleculemust be provided in activated form, i.e. with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl proprionate (SPA), succinimidy carboxymethylate(SCM), benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS),aldehyde, nitrophenylcarbonate (NPC), and tresylate (TRES)). Suitablyactivated polymer molecules are commercially available, e.g. fromShearwater Polymers, Inc., Huntsville, Ala., USA. Alternatively, thepolymer molecules can be activated by conventional methods known in theart, e.g. as disclosed in WO 90/13540. Specific examples of activatedlinear or branched polymer molecules for use in the present inventionare described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogue(Functionalized Biocompatible Polymers for Research and pharmaceuticals,Polyethylene Glycol and Derivatives, incorporated herein by reference).Specific examples of activated PEG polymers include the following linearPEGs: NHS-PEG (e.g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG,SG-PEG, and SCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG,CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branchedPEGs such as PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 andU.S. Pat. No. 5,643,575, both of which references are incorporatedherein by reference. Furthermore, the following publications,incorporated herein by reference, disclose useful polymer moleculesand/or PEGylation chemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No.5,476,653, WO 97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502,U.S. Pat. No. 5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No.5,219,564, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131,U.S. Pat. No. 5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No.5,629,384, WO 96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat.No. 5,516,673, EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400472, EP 183 503 and EP 154 316.

Specific examples of activated PEG polymers particularly preferred forcoupling to cysteine residues, include the following linear PEGs:vinylsulfone-PEG (VS-PEG), preferably vinylsulfone-mPEG (VS-mPEG);maleimide-PEG (MAL-PEG), preferably maleimide-mPEG (MAL-mPEG) andorthopyridyl-disulfide-PEG (OPSS-PEG), preferablyorthopyridyl-disulfide-mPEG (OPSS-mPEG). Typically, such PEG or MPEGpolymers will have a size of about 5 kDa, about 10 kD, about 12 kDa orabout 20 kDa.

The conjugation of the polypeptide variant and the activated polymermolecules is conducted by use of any conventional method, e.g. asdescribed in the following references (which also describe suitablemethods for activation of polymer molecules): Harris and Zalipsky, eds.,Poly(ethylene glycol) Chemistry and Biological Applications, AZC,Washington; R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N. Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y.). For PEGylation to cysteine residues (see above)the IFNG variant is usually treated with a reducing agent, such asdithiothreitol (DDT) prior to PEGylation. The reducing agent issubsequently removed by any conventional method, such as by desalting.Conjugation of PEG to a cysteine residue typically takes place in asuitable buffer at pH 6-9 at temperatures varying from 4° C. to 25° C.for periods up to 16 hours.

The skilled person will be aware that the activation method and/orconjugation chemistry to be used depends on the attachment group(s) ofthe polypeptide variant as well as the functional groups of the polymer(e.g. being amino, hydroxyl, carboxyl, aldehyde or sulfydryl). ThePEGylation may be directed towards conjugation to all availableattachment groups on the polypeptide variant (i.e. such attachmentgroups that are exposed at the surface of the polypeptide) or may bedirected towards specific attachment groups, e.g. the N-terminal aminogroup (U.S. Pat. No. 5,985,265). Furthermore, the conjugation may beachieved in one step or in a stepwise manner (e.g. as described in WO99/55377).

It will be understood that the PEGylation is designed so as to producethe optimal molecule with respect to the number of PEG moleculesattached, the size and form (e.g. whether they are linear or branched)of such molecules, and where in the polypeptide variant such moleculesare attached. For instance, the molecular weight of the polymer to beused may be chosen on the basis of the desired effect to be achieved.For instance, if the primary purpose of the conjugation is to achieve aconjugate having a high molecular weight (e.g. to reduce renalclearance) it is usually desirable to conjugate as few high molecularweight-polymer molecules as possible to obtain the desired molecularweight. When a high degree of epitope shielding is desirable this may beobtained by use of a sufficiently high number of low molecular weightpolymer (e.g. with a molecular weight of about 5,000 Da) to effectivelyshield all or most epitopes of the polypeptide. For instance, 2-8, suchas 3-6, of such polymers may be used.

In connection with conjugation to only a single attachment group on theprotein (as described in U.S. Pat. No. 5,985,265), it may beadvantageous that the polymer molecule, which may be linear or branched,has a high molecular weight, e.g. about 20 kDa.

Normally, the polymer conjugation is performed under conditions aimingat reacting all available polymer attachment groups with polymermolecules. Typically, the molar ratio of activated polymer molecules topolypeptide variant is 1000-1, in particular 200-1, e.g. 100-1, such as10-1 or 5-1 in order to obtain optimal reaction. However, also equimolarratios may be used.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide variant through a linker. Suitable linkersare well known to the skilled person. A preferred example is cyanuricchloride (Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581;U.S. Pat. No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym.Chem. Ed., 24, 375-378.

Subsequent to the conjugation residual activated polymer molecules areblocked according to methods known in the art, e.g. by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules removed by a suitable method.

Coupling to a Sugar Moiety

The coupling of a sugar moiety may take place in vivo or in vitro. Inorder to achieve in vivo glycosylation of a polypeptide with IFNGactivity, which have been modified so as to introduce one or more invivo glycosylation sites (see the section “IFNG variants of theinvention wherein the non-polypeptide moiety is a sugar moiety), thenucleotide sequence encoding the polypeptide must be inserted in aglycosylating, eukaryotic expression host. The expression host cell maybe selected from fungal (filamentous fungal or yeast), insect or animalcells or from transgenic plant cells. Furthermore, the glycosylation maybe achieved in the human body when using a nucleotide sequence encodingthe polypeptide variant of the invention in gene therapy. In oneembodiment the host cell is a mammalian cell, such as an CHO cell, BHKor HEK cell, e.g. HEK293, or an insect cell, such as an SF9 cell, or ayeast cell, e.g. Saccharomyces cerevisiae, Pichia pastoris or any othersuitable glycosylating host, e.g. as described further below.Optionally, sugar moieties attached to the IFNG polypeptide variant byin vivo glycosylation are further modified by use ofglycosyltransferases, e.g. using the glycoAdvance™ technology marketedby Neose, Horsham, Pa., USA. Thereby, it is possible to, e.g., increasethe sialyation of the glycosylated IFNG polypeptide variant followingexpression and in vivo glycosylation by CHO cells.

Covalent in vitro coupling of glycosides to amino acid residues of IFNGpolypepeptides may be used to modify or increase the number or profileof carbohydrate substituents. Depending on the coupling mode used, thesugar(s) may be attached to a) arginine and histidine, b) free carboxylgroups, c) free sulfhydryl groups such as those of cysteine, d) freehydroxyl groups such as those of serine, threonine, tyrosine orhydroxyproline, e) aromatic residues such as those of phenylalanine ortryptophan or f) the amide group of glutamine. These amino acid residuesconstitute examples of attachment groups for a sugar moiety, which maybe introduced and/or removed in the IFNG polypeptide. Suitable methodsof in vitro coupling are described, for example, in WO 87/05330 and inAplin et al., CRC Crit Rev. Biochem., pp. 259-306, 1981. The in vitrocoupling of sugar moieties or PEG to protein- and peptide-boundGln-residues can also be carried out by transglutaminases (TGases), e.g.as described by Sato et al., 1996 Biochemistry 35, 13072-13080 or in EP725145.

Coupling to an Organic Derivatizing Agent

Covalent modification of the IFNG polypeptide variant may be performedby reacting (an) attachment group(s) of the polypeptide variant with anorganic derivatizing agent. Suitable derivatizing agents and methods arewell known in the art. For example, cysteinyl residues most commonly arereacted with α-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are derivatized by reaction withdiethylpyrocarbonateat pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0. Lysinyl and amino terminal residues are reacted with succinicor other carboxylic acid anhydrides. Derivatization with these agentshas the effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate. Arginyl residues are modified by reaction with one orseveral conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginine residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine guanidino group. Carboxyl side groups (aspartyl orglutamyl) are selectively modified by reaction with carbodiimides(R—N═C═N—R′), where R and R′ are different alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Blocking of Functional Site

It has been reported that excessive polymer conjugation can lead to aloss of activity of the polypeptide to which the polymer is conjugated.This problem can be eliminated, e.g., by removal of attachment groupslocated at the functional site or by blocking the functional site priorto conjugation. These latter strategies constitute further embodimentsof the invention, the first strategy being exemplified further above,e.g. by removal of lysine residues which may be located close to thefunctional site and/or by introducing cysteine residues and/or in vivoglycosylation sites at positions not interfering with functional sites.

More specifically, according to the second strategy the conjugationbetween the polypeptide variant and the non-polypeptide moiety isconducted under conditions where the functional site of the IFNGpolypeptide variant is blocked by a helper molecule capable of bindingto the functional site of the polypeptide variant. Preferably, thehelper molecule is one, which specifically recognizes a functional siteof the polypeptide variant, such as a receptor. Alternatively, thehelper molecule may be an antibody, in particular a monoclonal antibodyrecognizing the polypeptide variant. In particular, the helper moleculemay be a neutralizing monoclonal antibody.

The polypeptide variant is then allowed to interact with the helpermolecule before effecting conjugation. This ensures that the functionalsite of the polypeptide variant is shielded or protected andconsequently unavailable for derivatization by the non-polypeptidemoiety such, as a polymer. Following its elution from the helpermolecule, the conjugate between the non-polypeptide moiety and thepolypeptide variant can be recovered with at least a partially preservedfunctional site.

The subsequent conjugation of the polypeptide variant having a blockedfunctional site to a polymer, a lipophilic compound, a sugar moiety, anorganic derivatizing agent or any other compound is conducted in thenormal way, e.g. as described in the sections above entitled“Conjugation to . . . ”.

In a further embodiment the helper molecule is first covalently linkedto a solid phase such as column packing materials, for instance Sephadexor agarose beads, or a surface, e.g. reaction vessel. Subsequently, thepolypeptide variant is loaded onto the column material carrying thehelper molecule and conjugation carried out according to methods knownin the art, e.g. as described in the sections above entitled“Conjugation to . . . ”. This procedure allows the conjugatedpolypeptide variant to be separated from the helper molecule by elution.The conjugated polypeptide variant is eluted by conventional techniquesunder physico-chemical conditions that do not lead to a substantivedegradation of the conjugated polypeptide variant. The fluid phasecontaining the conjugated polypeptide variant is separated from thesolid phase to which the helper molecule remains covalently linked. Theseparation can be achieved in other ways: For instance, the helpermolecule may be derivatized with a second molecule (e.g. biotin) thatcan be recognized by a specific binder (e.g. streptavidin). The specificbinder may be linked to a solid phase thereby allowing the separation ofthe conjugated polypeptide variant from the helper molecule-secondmolecule complex through passage over a second helper-solid phase columnwhich will retain, upon subsequent elution, the helper molecule-secondmolecule complex, but not the conjugated polypeptide variant. Theconjugated polypeptide variant may be released from the helper moleculein any appropriate fashion. De-protection may be achieved by providingconditions in which the helper molecule dissociates from the functionalsite of the polypeptide variant to which it is bound. For instance, acomplex between an antibody to which a polymer is conjugated and ananti-idiotypic antibody can be dissociated by adjusting the pH to anacid or alkaline pH.

Conjugation of a Tagged Polypeptide Variant

In an alternative embodiment the IFNG polypeptide variant is expressed,as a fusion protein, with a tag, i.e. an amino acid sequence or peptidestretch made up of typically 1-30, such as 1-20 amino acid residues.Besides allowing for fast and easy purification, the tag is a convenienttool for achieving conjugation between the tagged IFNG polypeptidevariant and the non-polypeptide moiety. In particular, the tag may beused for achieving conjugation in microtiter plates or other carriers,such as paramagnetic beads, to which the tagged polypeptide can beimmobilised via the tag. The conjugation to the tagged IFNG polypeptidevariantin, e.g., microtiter plates has the advantage that the taggedpolypeptide variant can be immobilised in the microtiter plates directlyfrom the culture broth (in principle without any purification) andsubjected to conjugation. Thereby, the total number of process steps(from expression to conjugation) can be reduced. Furthermore, the tagmay function as a spacer molecule ensuring an improved accessibility tothe immobilised polypeptide variant to be conjugated. The conjugationusing a tagged polypeptide variant may be to any of the non-polypeptidemoieties disclosed herein, e.g. to a polymer molecule such as PEG.

The identity of the specific tag to be used is not critical as long asthe tag is capable of being expressed with the polypeptide variant andis capable of being immobilised on a suitable surface or carriermaterial. A number of suitable tags are commercially available, e.g.from Unizyme Laboratories, Denmark. For instance, the tag may any of thefollowing sequences: (SEQ ID NO:35) His-His-His-His-His-His, (SEQ IDNO:36) Met-Lys-His-His-His-His-His-His, (SEQ ID NO:37)Met-Lys-His-His-Ala-His-His-Gln-His-His, (SEQ ID NO:38Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln- His-Gln,(all available from Unizyme Laboratories, Denmark)

or any of the following: EQKLI SEEDL (SEQ ID NO:39) (a C-terminal tagdescribed in Mol. Cell. Biol. 5:3610-16, 1985), DYKDDDDK (SEQ ID NO:40)(a C- or N-terminal tag), YPYDVPDYA (SEQ ID NO:41)

Antibodies against the above tags are commercially available, e.g. fromADI, Aves Lab and Research Diagnostics.

The subsequent cleavage of the tag from the polypeptide may be achievedby use of commercially available enzymes.

Methods of Preparing an IFNG Polypeptide Variant of the Invention

The IFNG polypeptide variant may be produced by any suitable methodknown in the art. Such methods include constructing a nucleotidesequence encoding the polypeptide variant and expressing the sequence ina suitable transformed or transfected host. However, polypeptidevariants of the invention may be produced, albeit less efficiently, bychemical synthesis or a combination of chemical synthesis or acombination of chemical synthesis and recombinant DNA technology.

The nucleotide sequence of the invention encoding an IFNG polypeptidevariant (in monomer or single chain form) may be constructed byisolating or synthesizing a nucleotide sequence encoding the parentIFNG, such as huIFNG with the amino acid sequence SEQ ID NO:17 or afragment thereof, and then changing the nucleotide sequence so as toeffect introduction (i.e. insertion or substitution) or deletion (i.e.removal or substitution) of the relevant amino acid residue(s).

The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with well-known methods, see, e.g., Mark etal., “Site-specific Mutagenesis of the Human Fibroblast InterferonGene”, Proc. Natl. Acad. Sci. USA, 81, pp. 5662-66 (1984); and U.S. Pat.No. 4,588,585.

Alternatively, the nucleotide sequence is prepared by chemicalsynthesis, e.g. by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide variant, and preferably selecting those codons thatare favored in the host cell in which the recombinant polypeptidevariant will be produced. For example, several small oligonucleotidescoding for portions of the desired polypeptide variant may besynthesized and assembled by PCR, ligation or ligation chain reaction(LCR). The individual oligonucleotides typically contain 5′ or 3′overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleotide sequence encoding the polypeptide variant isinserted into a recombinant vector and operably linked to controlsequences necessary for expression of the IFNG polypeptide variant inthe desired transformed host cell.

It should of course be understood that not all vectors and expressioncontrol sequences function equally well to express the nucleotidesequence encoding an IFNG polypeptide variant described herein. Neitherwill all hosts function equally well with the same expression system.However, one of skill in the art may make a selection among thesevectors, expression control sequences and hosts without undueexperimentation. For example, in selecting a vector, the host must beconsidered because the vector must replicate in it or be able tointegrate into the chromosome. The vector's copy number, the ability tocontrol that copy number, and the expression of any other proteinsencoded by the vector, such as antibiotic markers, should also beconsidered. In selecting an expression control sequence, a variety offactors should also be considered. These include, for example, therelative strength of the sequence, its controllability, and itscompatibility with the nucleotide sequence encoding the polypeptidevariant, particularly as regards potential secondary structures. Hostsshould be selected by consideration of their compatibility with thechosen vector, the toxicity of the product coded for by the nucleotidesequence, their secretion characteristics, their ability to fold thepolypeptide correctly, their fermentation or culture requirements, andthe ease of purification of the products coded for by the nucleotidesequence.

The recombinant vector may be an autonomously replicating vector, i.e. avector, which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector is one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector, in which the nucleotidesequence encoding the IFNG polypeptide variant is operably linked toadditional segments required for transcription of the nucleotidesequence. The vector is typically derived from plasmid or viral DNA. Anumber of suitable expression vectors for expression in the host cellsmentioned herein are commercially available or described in theliterature. Useful expression vectors for eukaryotic hosts, include, forexample, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectorsare, e.g., pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) andpCI-neo (Stratagene, La Jola, Calif., USA). Useful expression vectorsfor bacterial hosts include known bacterial plasmids, such as plasmidsfrom E. coli, including pBR322, pET3a and pET12a (both from NovagenInc., WI, USA), wider host range plasmids, such as RP4, phage DNAs,e.g., the numerous derivatives of phage lambda, e.g., NM989, and otherDNA phages, such as M13 and filamentous single stranded DNA phages.Useful expression vectors for yeast cells include the 2μ plasmid andderivatives thereof, the POT1 vector (U.S. Pat. No. 4,931,373), thepJSO37 vector described in (Okkels, Ann. New York Acad. Sci. 782,202-207, 1996) and pPICZ A, B or C (Invitrogen). Useful vectors forinsect cells include pVL941, pBG311 (Cate et al., “Isolation of theBovine and Human Genes for Mullerian Inhibiting Substance And Expressionof the Human Gene In Animal Cells”, Cell, 45, pp. 685-98 (1986),pBluebac 4.5 and pMelbac (both available from Invitrogen).

Other vectors for use in this invention include those that allow thenucleotide sequence encoding the IFNG polypeptide variant to beamplified in copy number. Such amplifiable vectors are well known in theart. They include, for example, vectors able to be amplified by DHFRamplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman andSharp, “Construction Of A Modular Dihydrafolate Reductase cDNA Gene:Analysis Of Signals Utilized For Efficient Expression”, Mol. Cell.Biol., 2, pp. 1304-19 (1982)) and glutamine synthetase (“GS”)amplification (see, e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

The recombinant vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. An example of such asequence (when the host cell is a mammalian cell) is the SV40 origin ofreplication. When the host cell is a yeast cell, suitable sequencesenabling the vector to replicate are the yeast plasmid 2μ replicationgenes REP 1-3 and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell, such as the genecoding for dihydrofolate reductase (DHFR) or the Schizosaccharomycespombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130),or one which confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. Forfilamentous fungi, selectable markers include amdS, pyrG, arcB, niaD,sC.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofthe IFNG polypeptide variant. Each control sequence may be native orforeign to the nucleic acid sequence encoding the polypeptide variant.Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, enhancer orupstream activating sequence, signal peptide sequence, and transcriptionterminator. At a minimum, the control sequences include a promoter.

A wide variety of expression control sequences may be used in thepresent invention. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription inmammalian cells include the early and late promoters of SV40 andadenovirus, e.g. the adenovirus 2 major late promoter, the MT-1(metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus E1bregion polyadenylation signals and the Kozak consensus sequence (Kozak,M. J Mol Biol 1987 Aug. 20; 196(4):947-50).

In order to improve expression in mammalian cells a synthetic intron maybe inserted in the 5′ untranslated region of the nucleotide sequenceencoding the IFNG polypeptide variant. An example of a synthetic intronis the synthetic intron from the plasmid pCI-Neo (available from PromegaCorporation, WI, USA).

Examples of suitable control sequences for directing transcription ininsect cells include the polyhedrin promoter, the P10 promoter, theAutographa californica polyhedrosis virus basic protein promoter, thebaculovirus immediate early gene 1 promoter and the baculovirus 39Kdelayed-early gene promoter, and the SV40 polyadenylation sequence.

Examples of suitable control sequences for use in yeast host cellsinclude the promoters of the yeast α-mating system, the yeast triosephosphate isomerase (TPI) promoter, promoters from yeast glycolyticgenes or alcohol dehydogenase genes, the ADH2-4c promoter and theinducible GAL promoter.

Examples of suitable control sequences for use in filamentous fungalhost cells include the ADH3 promoter and terminator, a promoter derivedfrom the genes encoding Aspergillus oryzae TAKA amylase triose phosphateisomerase or alkaline protease, an A. niger α-amylase, A. niger or A.nidulans glucoamylase, A. nidulans acetamidase, Rhizomucor mieheiaspartic proteinase or lipase, the TPII terminator and the ADH3terminator.

Examples of suitable control sequences for use in bacterial host cellsinclude promoters of the lac system, the trp system, the TAC or TRCsystem and the major promoter regions of phage lambda.

The nucleotide sequence of the invention, whether prepared bysite-directed mutagenesis, synthesis or other methods, may or may notalso include a nucleotide sequence that encode a signal peptide. Thesignal peptide is present when the polypeptide is to be secreted fromthe cells in which it is expressed. Such signal peptide, if present,should be one recognized by the cell chosen for expression of thepolypeptide variant. The signal peptide may be homologous (e.g. be thatnormally associated with huIFNG) or heterologous (i.e. originating fromanother source than huIFNG) to the polypeptide or may be homologous orheterologous to the host cell, i.e. be a signal peptide normallyexpressed from the host cell or one which is not normally expressed fromthe host cell. Accordingly, the signal peptide may be prokaryotic, e.g.derived from a bacterium such as E. coli, or eukaryotic, e.g. derivedfrom a mammalian, or insect or yeast cell.

The presence or absence of a signal peptide will, e.g., depend on theexpression host cell used for the production of the polypeptide variant,the protein to be expressed (whether it is an intracellular orintracellular protein) and whether it is desirable to obtain secretion.For use in filamentous fungi, the signal peptide may conveniently bederived from a gene encoding an Aspergillus sp. amylase or glucoamylase,a gene encoding a Rhizomucor miehei lipase or protease or a Humicolalanuginosa lipase. The signal peptide is preferably derived from a geneencoding A. oryzae TAKA amylase, A. niger neutral α-amylase, A. nigeracid-stable amylase, or A. niger glucoamylase. For use in insect cells,the signal peptide may conveniently be derived from an insect gene (cf.WO 90/05783), such as the lepidopteran Manduca sexta adipokinetichormone precursor, (cf U.S. Pat. No. 5,023,328), the honeybee melittin(Invitrogen), ecdysteroid UDPglucosyltransferase (egt) (Murphy et al.,Protein Expression and Purification 4, 349-357 (1993) or humanpancreatic lipase (hpl) (Methods in Enzymology 284, pp. 262-272, 1997).

A preferred signal peptide for use in mammalian cells is that of huIFNGor the murine Ig kappa light chain signal peptide (Coloma, M (1992) J.Imm. Methods 152:89-104). For use in yeast cells suitable signalpeptides have been found to be the α-factor signal peptide from S.cereviciae. (cf. U.S. Pat. No. 4,870,008), the signal peptide of mousesalivary amylase (cf. 0. Hagenbuchle et al., Nature 289, 1981, pp.643-646), a modified carboxypeptidase signal peptide (cf. L. A. Valls etal., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO87/02670), and the yeast aspartic protease 3 (YAP3) signal peptide (cf.M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

Any suitable host may be used to produce the IFNG polypeptide variant,including bacteria, fungi (including yeasts), plant, insect, mammal, orother appropriate animal cells or cell lines, as well as transgenicanimals or plants. Examples of bacterial host cells include grampositivebacteria such as strains of Bacillus, e.g. B. brevis or B. subtilis,Pseudomonas or Streptomyces, or gramnegative bacteria, such as strainsof E. coli. The introduction of a vector into a bacterial host cell may,for instance, be effected by protoplast transformation (see, e.g., Changand Cohen, 1979, Molecular General Genetics 168: 111-115), usingcompetent cells (see, e.g., Young and Spizizen, 1961, Journal ofBacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journalof Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawaand Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g.,Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

Examples of suitable filamentous fungal host cells include strains ofAspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium orTrichoderma. Fungal cells may be transformed by a process involvingprotoplast formation, transformation of the protoplasts, andregeneration of the cell wall in a manner known per se. Suitableprocedures for transformation of Aspergillus host cells are described inEP 238 023 and U.S. Pat. No. 5,679,543. Suitable methods fortransforming Fusarium species are described by Malardier et al., 1989,Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using theprocedures described by Becker and Guarente, In Abelson, J. N. andSimon, M. I., editors, Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., NewYork; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen etal., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Examples of suitable yeast host cells include strains of Saccharomyces,e.g. S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia, such asP. pastoris or P. methanolica, Hansenula, such as H. Polymorpha orYarrowia. Methods for transforming yeast cells with heterologous DNA andproducing heterologous polypeptides therefrom are disclosed by ClontechLaboratories, Inc, Palo Alto, Calif., USA (in the product protocol forthe Yeastmaker™ Yeast Tranformation System Kit), and by Reeves et al.,FEMS Microbiology Letters 99 (1992) 193-198, Manivasakam and Schiestl,Nucleic Acids Research, 1993, Vol. 21, No. 18, pp. 4414-4415 and Ganevaet al., FEMS Microbiology Letters 121 (1994) 159-164.

Examples of suitable insect host cells include a Lepidoptora cell line,such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells(High Five) (U.S. Pat. No. 5,077,214). Transformation of insect cellsand production of heterologous polypeptides therein may be performed asdescribed by Invitrogen.

Examples of suitable mammalian host cells include Chinese hamster ovary(CHO) cell lines, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cell lines(COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells(e.g. NS/O), Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 orATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573)), as well asplant cells in tissue culture. Additional suitable cell lines are knownin the art and available from public depositories such as the AmericanType Culture Collection, Rockville, Md. Also, the mammalian cell, suchas a CHO cell, may be modified to express sialyltransferase, e.g.1,6-sialyltransferase, e.g. as described in U.S. Pat. No. 5,047,335, inorder to provide improved glycosylation of the IFNG polypeptide variant.

Methods for introducing exogenous DNA into mammalian host cells includecalcium phosphate-mediated transfection, electroporation, DEAE-dextranmediated transfection, liposome-mediated transfection, viral vectors andthe transfection method described by Life Technologies Ltd, Paisley, UKusing Lipofectamin 2000. These methods are well known in the art ande.g. described by Ausbel et al. (eds.), 1996, Current Protocols inMolecular Biology, John Wiley & Sons, New York, USA. The cultivation ofmammalian cells are conducted according to established methods, e.g. asdisclosed in (Animal Cell Biotechnology, Methods and Protocols, Editedby Nigel Jenkins, 1999, Human Press Inc, Totowa, N.J., USA and HarrisonM A and Rae I F, General Techniques of Cell Culture, CambridgeUniversity Press 1997).

In order to produce a glycosylated polypeptide a eukaryotic host cell,e.g. of the type mentioned above, is used.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide variant using methods known in the art. For example, thecell may be cultivated by shake flask cultivation, small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentersperformed in a suitable medium and under conditions allowing thepolypeptide variant to be expressed and/or isolated. The cultivationtakes place in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide variant is secreted intothe nutrient medium, the polypeptide variant can be recovered directlyfrom the medium. If the polypeptide variant is not secreted, it can berecovered from cell lysates.

The resulting polypeptide variant may be recovered by methods known inthe art. For example, the polypeptide variant may be recovered from thenutrient medium by conventional procedures including, but not limitedto, centrifugation, filtration, extraction, spray drying, evaporation,or precipitation.

The polypeptide variants may be purified by a variety of proceduresknown in the art including, but not limited to, chromatography (e.g.,ion exchange, affinity, hydrophobic, chromatofocusing, and sizeexclusion), electrophoretic procedures (e.g., preparative isoelectricfocusing), differential solubility (e.g., ammonium sulfateprecipitation), HPLC, or extraction (see, e.g., Protein Purification,J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).Specific methods for purifying polypeptides exhibiting IFNG activity aredisclosed in EP 0 110 044 and unexamined Japanese patent application No.186995/84.

The biological activity of the IFNG polypeptide variant can be assayedby any suitable method known in the art. Such assays include antibodyneutralization of antiviral activity, induction of protein kinase,oligoadenylate 2,5-A synthetase or phosphodiesterase activities, asdescribed in EP 0 041 313 B1. Such assays also include immunomodulatoryassays (see, e.g., U.S. Pat. No. 4,753,795), growth inhibition assays,and measurement of binding to cells that express interferon receptors.Specific assays are described in the Materials and Methods sectionherein.

Pharmaceutical Compositions and Uses Thereof

Furthermore, the present invention relates to improved methods oftreating or preventing, in particular, inflammatory diseases, e.g.interstitial lung diseases, such as idiopathic pulmonary fibrosis, butalso granulomatous diseases; cancer, in particular ovarian cancer;infections such as pulmonary atypical mycobacterial infections; bonedisorders (e.g. a bone metabolism disorder so as malignantosteopetrosis); autoimmune diseases such as rheumatoid arthritis; aswell as other diseases such as multiresistent tuberculosis; cryptococcalmeningitis; cystic fibrosis and liver fibrosis, in particular liverfibrosis secondary to hepatitis C, said method comprising administeringto a mammal, in particular a human being, in need thereof an effectiveamount of a polypeptide variant of the invention or a composition of theinvention; the key advantages being less frequent and/or less intrusiveadministration of more efficient therapy, and optionally a lower risk ofimmune reactions with the therapeutically active compound(s).

The molecule of the invention is preferably administered in acomposition including a pharmaceutically acceptable carrier orexcipient. “Pharmaceutically acceptable” means a carrier or excipientthat does not cause any untoward effects in patients to whom it isadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]).

The molecules of the invention can be used “as is” and/or in a salt formthereof. Suitable salts include, but are not limited to, salts withalkali metals or alkaline earth metals, such as sodium, potassium,calcium and magnesium, as well as e.g. zinc salts. These salts orcomplexes may by present as a crystalline and/or amorphous structure.

The variant of the invention is administered at a dose approximatelyparalleling that employed in therapy with known commercial preparationsof IFNG such as Actimmune® or as specified in EP 0 795 332. The exactdose to be administered depends on the circumstances. Normally, the doseshould be capable of preventing or lessening the severity or spread ofthe condition or indication being treated. It will be apparent to thoseof skill in the art that an effective amount of variant or compositionof the invention depends, inter alia, upon the disease, the dose, theadministration schedule, whether the polypeptide variant or compositionis administered alone or in conjunction with other therapeutic agents,the serum half-life of the compositions, and the general health of thepatient.

The present invention also relates to an IFNG polypeptide variantaccording to the present invention, or a pharmaceutical compositionaccording to the present invention, for use as a medicament.

Furthermore, the invention also relates to the use of i) an IFNG variantaccording to the present invention, or ii) a pharmaceutical compositionof the invention, for the manufacture of a medicament, a pharmaceuticalcomposition or a kit-of-parts for the treatment of diseases selectedfrom the group consisting of inflammatory diseases, such as interstitiallung diseases, in particular idiopathic pulmonary fibrosis; cancer, inparticular ovarian cancer; infections, such as pulmonary atypicalmycobacterial infections; bone disorders (e.g. a bone metabolismdisorder so as malignant osteopetrosis); granulomatous diseases;autoimmune diseases such as rheumatoid arthritis; multiresistenttuberculosis; cryptococcal meningitis; cystic fibrosis and liverfibrosis, in particular liver fibrosis secondary to hepatitis C. Mostpreferably the disease is an interstitial lung disease, in particularidiopathic pulmonary fibrosis.

Also disclosed are improved means of delivering the molecules orpreparations, optionally additionally comprising glucocorticoids.

The preferred dosing is 1-4, more preferably 2-3, micrograms/kg patientweight of the polypeptide component per dose. The preferred dosing is100-350, more preferably 100-150 micrograms glucocorticoid/kg patientweight per dose.

The invention also relates to a kit of parts suitable for the treatmentof interstitial lung diseases comprising a first pharmaceuticalcomposition comprising the active components i) or ii) mentioned aboveand a second pharmaceutical composition comprising at least oneglucocorticoid, each optionally together with a pharmaceuticallyacceptable carrier and/or excipient.

The variant of the invention can be formulated into pharmaceuticalcompositions by well-known methods. Suitable formulations are describedby Remington's Pharmaceutical Sciences by E. W. Martin and U.S. Pat. No.5,183,746.

The pharmaceutical composition may be formulated in a variety of forms,including liquid, gel, lyophilized, powder, compressed solid, or anyother suitable form. The preferred form will depend upon the particularindication being treated and will be apparent to one of skill in theart.

The pharmaceutical composition may be administered orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner,e.g. using PowderJect or ProLease technology. The formulations can beadministered continuously by infusion, although bolus injection isacceptable, using techniques well known in the art, such as pumps orimplantation. In some instances the formulations may be directly appliedas a solution or spray. The preferred mode of administration will dependupon the particular indication being treated and will be apparent to oneof skill in the art.

The pharmaceutical composition of the invention may be administered inconjunction with other therapeutic agents. These agents may beincorporated as part of the same pharmaceutical composition or may beadministered separately from the polypeptide variant of the invention,either concurrently or in accordance with any other acceptable treatmentschedule. In addition, the polypeptide variant or pharmaceuticalcomposition of the invention may be used as an adjunct to othertherapies. In particular, combinations with glucocorticoids as describedin EP 0 795 332 are considered.

In a further aspect the invention relates to a method of treating amammal having circulating antibodies against huIFNG or rhuIFNG, whichmethod comprises administering an IFNG variant which has the bioactivityof IFNG and which does not react with said antibodies. The compound ispreferably a variant as described herein and the mammal is preferably ahuman being. The mammals to be treated may suffer from any of thediseases listed above for which IFNG is a useful treatment. Furthermore,the invention relates to a method of making a pharmaceutical product foruse in treatment of mammals having circulating antibodies against huIFNGor rhuIFNG, wherein an IFNG variant which has the bioactivity of IFNGand which does not react with such is formulated into an injectable orotherwise suitable formulation. The term “circulating antibodies” isintended to indicate autoantibodies formed in a mammal in response tohaving been treated with any of the commercially available IFNGpreparations.

Also contemplated is use of a nucleotide sequence encoding an IFNGpolypeptide variant of the invention in gene therapy applications. Inparticular, it may be of interest to use a nucleotide sequence encodingan IFNG polypeptide variant described in the section above entitled“IFNG variants of the invention wherein the non-polypeptide moiety is asugar moiety”. The glycosylation of the polypeptide variant is thusachieved during the course of the gene therapy, i.e. after expression ofthe nucleotide sequence in the human body.

Gene therapy applications contemplated include treatment of thosediseases in which the polypeptide variant is expected to provide aneffective therapy.

Local delivery of IFNG variant using gene therapy may provide thetherapeutic agent to the target area while avoiding potential toxicityproblems associated with non-specific administration.

Both in vitro and in vivo gene therapy methodologies are contemplated.

Several methods for transferring potentially therapeutic genes todefined cell populations are known. For further reference see, e.g.,Mulligan, “The Basic Science Of Gene Therapy”, Science, 260, pp. 926-31(1993). These methods include:

-   -   Direct gene transfer, e.g., as disclosed by Wolff et al.,        “Direct Gene transfer Into Mouse Muscle In vivo”, Science 247,        pp. 1465-68 (1990);    -   Liposome-mediated DNA transfer, e.g., as disclosed by Caplen et        al., “Liposome-mediated CFTR Gene Transfer to the Nasal        Epithelium Of Patients With Cystic Fibrosis” Nature Med., 3, pp.        39-46 (1995); Crystal, “The Gene As A Drug”, Nature Med., 1, pp.        15-17 (1995); Gao and Huang, “A Novel Cationic Liposome Reagent        For Efficient Transfection of Mammalian Cells”, Biochem. Biophys        Res. Comm., 179, pp. 280-85 (1991);    -   Retrovirus-mediated DNA transfer, e.g., as disclosed by Kay et        al., “In vivo Gene Therapy of Hemophilia B: Sustained Partial        Correction In Factor IX-Deficient Dogs”, Science, 262, pp.        117-19 (1993); Anderson, “Human Gene Therapy”, Science, 256, pp.        808-13(1992);    -   DNA Virus-mediated DNA transfer. Such DNA viruses include        adenoviruses (preferably Ad-2 or Ad-5 based vectors), herpes        viruses (preferably herpes simplex virus based vectors), and        parvoviruses (preferably “defective” or non-autonomous        parvovirus based vectors, more preferably adeno-associated virus        based vectors, most preferably AAV-2 based vectors). See, e.g.,        Ali et al., “The Use Of DNA Viruses as Vectors for Gene        Therapy”, Gene Therapy, 1, pp. 367-84 (1994); U.S. Pat. No.        4,797,368, and U.S. Pat. No. 5,139,941.        Parenterals

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

In case of parenterals, they are prepared for storage as lyophilizedformulations or aqueous solutions by mixing, as appropriate, thepolypeptide variant having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and/or othermiscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives may be added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g. benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers may be added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients, which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilize the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers(e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

In a preferred embodiment of the invention said pharmaceuticalcomposition comprises the i) IFNG variant of the invention, ii) abuffering agent, in particular a salt of an organic acid, capable ofmaintaining the pH between 5.0-6.5, iii) a stabilizer, in particular anorganic sugar or sugar alcohol, iv) a non-ionic surfactant, and v)sterile water. More particularly, the buffering agent is selected fromthe group consisting of acetate, succinate and citrate, the stabilizeris mannitol or sorbitol, the non-ionic surfactant is Tween®-20 orTween®-80. Preferably, the pharmaceutical composition does not includeany preservatives.

Sustained Release Preparations

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thevariant, the matrices having a suitable form such as a film ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Oral Administration

For oral administration, the pharmaceutical composition may be in solidor liquid form, e.g. in the form of a capsule, tablet, suspension,emulsion or solution. The pharmaceutical composition is preferably madein the form of a dosage unit containing a given amount of the activeingredient. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, butcan be determined by persons skilled in the art using routine methods.

Solid dosage forms for oral administration may include capsules,tablets, suppositories, powders and granules. In such solid dosageforms, the active compound may be admixed with at least one inertdiluent such as sucrose, lactose, or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances, e.g. lubricatingagents such as magnesium stearate. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

The variants may be admixed with adjuvants such as lactose, sucrose,starch powder, cellulose esters of alkanoic acids, stearic acid, talc,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulphuric acids, acacia, gelatin, sodium alginate,polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted orencapsulated for conventional administration. Alternatively, they may bedissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are well known in the pharmaceutical art. The carrieror diluent may include time delay material, such as glycerylmonostearate or glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants such as preservatives, stabilizers, wettingagents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhereherein.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants such as wetting agents,sweeteners, flavoring agents and perfuming agents.

for topical administration include liquid or semi-liquid prepa

etration through the skin (e.g., liniments, lotions, ointments, creams,or pastes) and drops suitable for administration to the eye, ear, ornose.

Pulmonary Delivery

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the polypeptide variant dissolved inwater at a concentration of, e.g., about 0.01 to 25 mg of variant per mLof solution, preferably about 0.1 to 10 mg/mL. The formulation may alsoinclude a buffer and a simple sugar (e.g., for protein stabilization andregulation of osmotic pressure), and/or human serum albumin ranging inconcentration from 0.1 to 10 mg/ml. Examples of buffers that may be usedare sodium acetate, citrate and glycine. Preferably, the buffer willhave a composition and molarity suitable to adjust the solution to a pHin the range of 3 to 9. Generally, buffer molarities of from 1 mM to 50mM are suitable for this purpose. Examples of sugars which can beutilized are lactose, maltose, mannitol, sorbitol, trehalose, andxylose, usually in amounts ranging from 1% to 10% by weight of theformulation.

The nebulizer formulation may also contain a surfactant to reduce orprevent surface induced aggregation of the protein caused by atomizationof the solution in forming the aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbitan fatty acid esters. Amounts will generallyrange between 0.001% and 4% by weight of the formulation. An especiallypreferred surfactant for purposes of this invention is polyoxyethylenesorbitan monooleate.

Specific formulations and methods of generating suitable dispersions ofliquid particles of the invention are described in WO 94/20069, U.S.Pat. No. 5,915,378, U.S. Pat. No. 5,960,792, U.S. Pat. No. 5,957,124,U.S. Pat. No. 5,934,272, U.S. Pat. No. 5,915,378, U.S. Pat. No.5,855,564, U.S. Pat. No. 5,826,570 and U.S. Pat. No. 5,522,385 which arehereby incorporated by reference.

Formulations for use with a metered dose inhaler device will generallycomprise a finely divided powder. This powder may be produced bylyophilizing and then milling a liquid variant formulation and may alsocontain a stabilizer such as human serum albumin (HSA). Typically, morethan 0.5% (w/w) HSA is added. Additionally, one or more sugars or sugaralcohols may be added to the preparation if necessary. Examples includelactose maltose, mannitol, sorbitol, sorbitose, trehalose, xylitol, andxylose. The amount added to the formulation can range from about 0.01 to200% (w/w), preferably from approximately 1 to 50%, of the variantpresent. Such formulations are then lyophilized and milled to thedesired particle size.

The properly sized particles are then suspended in a propellant with theaid of a surfactant. The propellant may be any conventional materialemployed for this purpose, such as a chloro fluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, includingtrichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant. Thismixture is then loaded into the delivery device. An example of acommercially available metered dose inhaler suitable for use in thepresent invention is the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C.

Formulations for powder inhalers will comprise a finely divided drypowder containing variant and may also include a bulking agent, such aslactose, sorbitol, sucrose, or mannitol in amounts which facilitatedispersal of the powder from the device, e.g., 50% to 90% by weight ofthe formulation. The particles of the powder shall have aerodynamicproperties in the lung corresponding to particles with a density ofabout 1 g/cm² having a median diameter less than 10 micrometers,preferably between 0.5 and 5 micrometers, most preferably of between 1.5and 3.5 micrometers. An example of a powder inhaler suitable for use inaccordance with the teachings herein is the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass.

The powders for these devices may be generated and/or delivered bymethods disclosed in U.S. Pat. No. 5,997,848, U.S. Pat. No. 5,993,783,U.S. Pat. No. 5,985,248, U.S. Pat. No. 5,976,574, U.S. Pat. No.5,922,354, U.S. Pat. No. 5,785,049 and U.S. Pat. No. 5,654,007.

Mechanical devices designed for pulmonary delivery of therapeuticproducts, include but are not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to those ofskill in the art. Specific examples of commercially available devicessuitable for the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.;the Ventolin metered dose inhaler, manufactured by Glaxo Inc., ResearchTriangle Park, N.C.; the Spinhaler powder inhaler, manufactured byFisons Corp., Bedford, Mass.; the “standing cloud” device of InhaleTherapeutic Systems, Inc., San Carlos, Calif.; the AIR inhalermanufactured by Alkermes, Cambridge, Mass.; and the AERx pulmonary drugdelivery system manufactured by Aradigm Corporation, Hayward, Calif.

The invention is further described in the following examples. Theexamples should not, in any manner, be understood as limiting thegenerality of the present specification and claims.

Materials and Methods

Materials

-   CHO-K1 cells (available from American Type Culture Collection (ATCC    #CCL-61)).-   HeLa cells (available from American Type Culture Collection (ATCC    #CCL-2)).-   ISRE-Luc was obtained from Stratagene, La Jolla USA.-   pCDNA 3.1/hygro was obtained from Invitrogen, Carlsbad USA.-   Restricion enzymes and polymerases were obtained from New England    Biolabs Inc., Beverly, USA.-   DMEM medium: Dulbecco's Modified Eagle Media (DMEM), 10% fetal    bovine serum and-   Hygromycin B were obtained from Life Technologies A/S, Copenhagen,    Denmark.-   LucLite substrate was obtained from Packard Bioscience, Groningen,    The Netherlands.-   TopCount luminometer was obtained from Packard Bioscience,    Groningen, The Netherlands.-   Biotinylated polyclonal anti-human IFNG antibody, BAF285, was    obtained available from R&D Systems Inc., Minneapolis, USA.-   Horse Radish Peroxidase-conjugated streptavidin, P0397, was obtained    from DAKO, Copenhagen, Denmark.-   TMB blotting reagent was obtained from KEM-EN-TEC, Copenhagen,    Denmark.    Methods    Interferon Assay Outline

It has previously been published that IFNG interacts with and activatesIFNG receptors on HeLa cells. Consequently, transcription is activatedat promoters containing an Interferon Stimulated Response Element(ISRE). It is thus possible to screen for agonists of interferonreceptors by use of an ISRE coupled luciferase reporter gene (ISRE-luc)placed in HeLa cells.

Primary Assay

HeLa cells are co-transfected with ISRE-Luc and pCDNA 3.1/hygro and foci(cell clones) are created by selection in DMEM media containingHygromycin B. Cell clones are screened for luciferase activity in thepresence or absence of IFNG. Those clones showing the highest ratio ofstimulated to unstimulated luciferase activity are used in furtherassays.

To screen polypeptide variants, 15,000 cells/well are seeded in 96 wellculture plates and incubated overnight in DMEM media. The next day thepolypeptide variants as well as a known standard are added to the cellsin various concentrations. Actimmune® was used as “known standard”; Avial of Actimmune® was diluted to 300 IU/ml in DMEM, 5% FBS and storedat −80° C. until use.

The plates are incubated for 6 hours at 37 C in a 5% CO₂ air atmosphereLucLite substrate (Packard Bioscience, Groningen, The Netherlands) issubsequently added to each well. Plates are sealed and luminescencemeasured on a TopCount luminometer (Packard) in SPC (single photoncounting) mode.

Each individual plate contains wells incubated with IFNG as a stimulatedcontrol and other wells containing normal media as an unstimulatedcontrol. The ratio between stimulated and unstimulated luciferaseactivity serves as an internal standard for both IFNG activity andexperiment-to-experiment variation. For each IFNG sample, the amount ofunits were calculated relative to the Actimmune® standard and given asAU.

Determination of Increased Degree of Glycosylation

To determine the various degrees of glycosylation of IFNG variantmonomers, a SDS-PAGE gel is run under standard conditions andtransferred to a nitrocellulose membrane. Western blotting is doneaccording to standard procedures using a biotinylated polyclonalanti-human IFNG antibody (BAF285 from R & D Systems) as primary antibodyand Horse Radish Peroxidase-conjugated streptavidin (P0397 from DAKO) assecondary antibody followed by staining with TMB blotting reagent(KEM-EN-TEC, Copenhagen, Denmark). The distribution of IFNG variantmonomers having varying degrees of glycosylation is made by visualinspection of the stained membrane.

Determination of AUC_(sc)

The AUC_(sc) is determined by one 200 μl bolus subcutaneousadministration of equal amount (on an activity basis) of the IFNGpolypeptide variant of the invention in rats.

For these experiments, female Sprag-Dawley rats, weiging between 220-260grams, are used. The IFNG polypeptide is formulated in sodium succinate(720 mg/l), mannitol 40 g/l), polysorbat 20 (100 mg/l) at pH 6.0.

Before subcutaneous administration, one blood sample is drawn in thetail-vein to ensure that no background IFNG activity can be detected.After administration, blood samples are withdrawn from the tail veinafter 10 min, 20 min, 40 min, 60 min, 120 min, 240 min, 480 min, 720min, 1440 min, 1620 min, 1920 min and 2880 min (sometimes also 3600min). Serum is prepared by letting the blood sample coagulate for 20 minat room temperature followed by centrifugation at 5000 g, 20 min at roomtemperature. The serum is then isolated and stored at −80° C. untildetermination of IFNG activity using the “Primary Assay” describedabove. It should be noted that when the amount of units were determinedin serum samples from PK studies in rats, the Actimmune® standard wasdiluted in DMEM, 5% FBS and 5% rat serum.

The amount of units in serum (AU/ml) against time (min) is then plottedand the AUC_(sc) is calculated using GraphPad Prism 3.01.

Similar experiments are performed on huIFNG in its glycosylated formand/or Actimmune® in order to assess the increase in AUC_(sc) of theIFNG polypeptide variant of the invention as compared to huIFNG in itsglycosylated form and/or Actimmune®.

Serum Half-Life

The serum half-life is determined by one 200 μl bolus intravenousadministration of equal amount (on an activity basis) of the IFNGpolypeptide variant of the invention in rats.

For these experiments, female Sprag-Dawley rats, weighing between220-260 grams, are used. The IFNG polypeptide variant is formulated insodium succinate (720 mg/l), mannitol 40 g/l), polysorbat 20 (100 mg/l)at pH 6.0.

Before intravenous administration, one blood sample is drawn in thetail-vein to ensure that no background IFNG activity can be detected.After administration in one tail vein, blood samples are withdrawn fromthe other tail vein after 5 min, 10 min, 20 min, 40 min, 60 min, 120min, 240 min, 480 min, 720 min, 1440 min, 1620 min, 1920 min and 2880min. Serum is prepared by letting the blood sample coagulate for 20 minat room temperature followed by centrifugation at 5000 g, 20 min at roomtemperature. The serum is then isolated and stored at −80° C. untildetermination of IFNG activity using the “Primary Assay” describedabove. It should be noted that when the amount of units were determinedin serum samples from PK studies in rats, the Actimmune® standard wasdiluted in DMEM, 5% FBS and 5% rat serum.

The amount of units in serum (AU/ml) against time (min) is then plottedand the serum half-life is calculated using WinNonLin Pro 3.3.

Similar experiments are performed on huIFNG in its glycosylated form,and/or Actimmune® in order to assess the increase in serum half-life ofthe IFNG polypeptide variant of the invention as compared to huIFNG inits glycosylated form and/or Actimmune®.

Identification of Surface Exposed Amino Acid Residues

Structures

Experimental 3D structures of huIFNG determined by X-ray crystallographyhave been reported by: Ealick et. al Science 252:698-702 (1991)reporting on the C-alpha trace of an IFNG homodimer. Walter et. al.Nature 376:230-235 (1995) reporting on the structure of an IFNGhomodimer in complex with two molecules of a soluble form of the IFNGreceptor. The coordinates of this structure have never been madepublicly available. Thiel et. al. Structure 8:927-936 (2000) reportingon the structure of an IFNG homodimer in complex with two molecules of asoluble form of the IFNG receptor having a third molecule of thereceptor in the structure not making interactions with the IFNGhomodimer.

Accessible Surface Area (ASA)

The computer program Access (B. Lee and F. M. Richards, J. Mol. Biol.55: 379-400 (1971)) version 2 (Copyright (c) 1983 Yale University) wasused to compute the accessible surface area (ASA) of the individualatoms in the structure. This method typically uses a probe-size of 1.4 Åand defines the Accessible Surface Area (ASA) as the area formed by thecentre of the probe. Prior to this calculation all water molecules,hydrogen atoms and other atoms not directly related to the protein areremoved from the coordinate set.

Fractional ASA of Side Chain

The fractional ASA of the side chain atoms is computed by division ofthe sum of the ASA of the atoms in the side chain with a valuerepresenting the ASA of the side chain atoms of that residue type in anextended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991)J. Mol. Biol.: 220,507-530. For this example the CA atom is regarded asa part of the side chain of Glycine residues but not for the remainingresidues. The following table are used as standard 100% ASA for the sidechain: Ala 69.23 Å² Arg 200.35 Å² Asn 106.25 Å² Asp 102.06 Å² Cys 96.69Å² Gln 140.58 Å² Glu 134.61 Å² Gly 32.28 Å² His 147.00 Å² Ile 137.91 Å²Leu 140.76 Å² Lys 162.50 Å² Met 156.08 Å² Phe 163.90 Å² Pro 119.65 Å²Ser 78.16 Å² Thr 101.67 Å² Trp 210.89 Å² Tyr 176.61 Å² Val 114.14 Å²

Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions.

Determining Distances Between Atoms:

The distance between atoms was determined using molecular graphicssoftware e.g. InsightII v. 98.0, MSI INC.

Determination of Receptor Binding Site:

The receptor-binding site is defined as comprising of all residueshaving their accessible surface area changed upon receptor binding. Thisis determined by at least two ASA calculations; one on the isolatedligand(s) in the ligand(s)/receptor(s) complex and one on the completeligand(s)/receptor(s) complex.

EXAMPLES Example 1 Determination of Surface-Exposed Amino Acid Residues

The X-ray structure used was of an IFNG homo-dimer in complex with twomolecules of a soluble form of the IFNG receptor having a third moleculeof the IFNG receptor in the structure not making interactions with theIFNG homodimer reported by Thiel et. al. Structure 8:927-936 (2000). Thestructure consists of the IFNG homodimer wherein the two molecules arelabeled A and B. For construction purposes there is an additionalmethionine placed before the IFNG sequence labeled M0 and the sequenceis C-terminally truancuted with ten residues (Q133 being the lastresidue in the constructed molecules). The M0 is removed from thestructure in all the calculations of this example. The structure of thetwo IFNG monomers has very weak electron density after residue 120 andresidues were only modeled until residue T126. Therefore, residuesS121-T126 were removed from the structure prior to the calculations inthis example. The two receptor fragments labeled C and D make directinteractions with the IFNG homodimer and a third receptor moleculelabeled E makes no contact with the IFNG homodimer and are not includedin these calculations.

Surface Exposure:

Performing fractional ASA calculations on the homodimer of molecules Aand B excluding M0 and S121-T126 in both molecules resulted in thefollowing residues having more than 25% of their side chain exposed tothe surface in at least one of the monomers: Q1, D2, P3, K6, E9, N10,K12, K13, Y14, N16, G18, H19, S20, D21, A23, D24, N25, G26, T27, G31,K34, N35, K37, E38, E39, S40, K55, K58, N59, K61, D62, D63, Q64, S65,Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84, N85, K86, K87,D90, E93, K94, N97, S99, T101, D102, L103, N104, H111, Q115, A118 andE119.

The following residues had more than 50% of their side chain exposed tothe surface in at least one of the monomers: Q1, D2, P3, K6, E9, N10,K13, N16, G18, H19, S20, D21, A23, D24, N25, G26, T27, G31, K34, K37,E38, E39, K55, K58, N59, D62, Q64, S65, K68, E71, E75, N83, S84, K86,K87, K94, N97, S99, T101, D102, L103, N104, Q115, A118, E119.

Performing fractional ASA calculations on the homodimer of molecules Aand B excluding M0 and S121-T126 in both molecules and including thereceptor molecules C and D resulted in the following residues had morethan 25% of their side chain exposed to the surface in at least one ofthe monomers: Q1, D2, P3, K6, E9, N0, K13, Y14, N16, G18, H19, D21, N25,G26, G31, K34, N35, K37, E38, E39, S40, K55, K58, N59, K61, D62, D63,Q64, S65, Q67, K68, E71, T72, K74, E75, N78, V79, K80, N83, S84, N85,K86, K87, D90, E93, K94, N97, S99, T101, D102, L103, N104, E119.

The following residues had more than 50% of their side chain exposed tothe surface in at least one of the monomers: P3, K6, N1, K13, N16, D21,N25, G26, G31, K34, K37, E38, E39, K55, K58, N59, D62, Q64, S65, K68,E71, E75, N83, S84, K86, K87, K94, N97, S99, T101, D102, L103 and N104.

All of the above positions are targets for modification in accordancewith the present invention.

Comparing the two lists, results in K12, S20, A23, D24, T27, H111, Q115and A118 being removed from the more than 25% side chain ASA list uponreceptor binding, and Q1, D2, E9, G18, H19, S20, A23, D24, T27, Q115,A118 and E119 being removed from the more than 50% side chain ASA listupon receptor binding.

Residues not determined in the structure are treated as fully surfaceexposed, i.e. residues S121, P122, A123, A124, K125, T126, G127, K128,R129, K130, R131, S132, Q133, M134, L135, F136, R137, G138, R139, R140,A141, S142, Q143. These residues also constitute separate targets forintroduction of attachment groups in accordance with the presentinvention (or may be viewed as belonging to the group of surface exposedamino acid residues, e.g. having more than 25% or more than 50% exposedside chains).

Example 2 Determination of the Receptor Binding Site

Performing ASA calculations as described above results in the followingresidues of the IFNG molecule having reduced ASA in at least one of themonomers in the complex as compared to the calculation on the isolateddimer: Q1, D2, Y4, V5, E9, K12, G18, H19, S20, D21, V22, A23, D24, N25,G26, T27, L30, K34, K37, K108, H111, E112, I114, Q115, A118, E119.

Example 3 Design of a Cassette for Expression of IFNG with OptimisedCodon Usage

The DNA sequence, GenBank accession number X13274, encompassing afull-length cDNA encoding mature huIFNG with its native signal peptide,was modified in order to facilitate high expression in CHO cells. Codonsof the huIFNG nucleotide sequence were modified by making a bias in thecodon usage towards the codons frequently used in homo sapiens.Subsequently, certain nucleotides in the sequence were substituted withothers in order to introduce recognition sites for DNA restrictionendonucleases. Primers were designed such that the gene could besynthesised.

The primers were assembled to the synthetic gene by one-step PCR usingthe platinum Pfx-polymerase kit (Life Technologies) and standardthree-step PCR cycling parameters. The assembled gene was amplified byPCR using the same conditions and has the sequence shown in SEQ IDNO:42. The synthesised gene was cloned into pcDNA3.1/hygro (InVitrogen)between the BamHI and the XbaI sites, resulting in pIGY-22.

pIGY-22 was transfected into CHO K1 cells by use of Lipofectaim2000(Life Technologies) as transfection agent. 24 hours later the culturemedium was harvested and assayed for IFNG activity and concentration byElisa. Using the Primary assay described herein, an activity of 1.4×10⁷AU/ml was obtained.

Example 4 Site Directed Mutagenesis

Generation of Glycosylation Variants

To introduce mutations in IFNG, oligonucleotides were designed in such away that PCR-generated changes could be introduced in the expressionplasmid (pIGY-22) by classical two-step PCR.

Two vector primers were used together with specific mutation primers:ADJ013: 5′-GATGGCTGGCAACTAGAAG-3′ (antisense downstream vector primer)(SEQ ID NO:43) and ADJ014: 5′-TGTACGGTGGGAGGTCTAT-3′ (SEQ ID NO:44)(sense upstream vector primer)

The S99T variant was generated by classical two-step PCR, using ADJ013and ADJ014 as vector primers, ADJ093(5′-GTTCAGGTCTGTCACGGTGTAATTGGTCAG-CTT-3′) (SEQ ID NO:45) and ADJ094(5′-AAGCTGACCAATTACACCGTGACAGA-CCTGAAC-3′) (SEQ ID NO:46) as mutationprimers, and pIGY-22 as template. The 447 bp PCR product was subclonedinto pcDNA3.1/Hygro (InVitrogen) using BamHI and XbaI, leading toplasmid pIGY-48.

pIGY-48 was transfected into CHO K1 cells by use of Lipofectaim2000(Life Technologies) as transfection agent. 24 hours later the culturemedium was harvested and assayed for IFNG activity. Using the Primaryassay described herein, the following activity was obtained: 5.1×10⁶AU/ml.

The E38N+S40T+S99T variant was generated by classical two-step PCR,using ADJ013 and ADJ014 as vector primers, ADJ091(5′-CATGATCTTCCGATCGGTCTC-GTTCTTCCAATT-3′) (SEQ ID NO:47) and ADJ092(5′-AATTGGAAGAACGAGACC-GATCGGAAGATCATG-3′) (SEQ ID NO:48) as mutationprimers, and pIGY-48 as template. The 447 bp PCR product was subclonedinto pcDNA3.1/Hygro (InVitrogen) using BamHI and XbaI, leading toplasmid pIGY-54.

pIGY-54 was transfected into CHO K1 cells by use of Lipofectaim2000(Life Technologies) as transfection agent. 24 hours later the culturemedium was harvested and assayed for IFNG activity. Using the Primaryassay described herein, an activity of 1.3×10⁷ AU/ml was obtained.

Using similar standard techniques as described above, a number offull-length IFNG glycosylation variants were prepared. These variantsare compiled in Table 1 below.

Generation of C-Terminally Truncated IFNG Variants

C-terminally truncated INFG variants, containing a stop codonimmediately downstream of the codon for Leu135, were generated byone-step PCR using pIGY-22, pIGY-48 and pIGY-54 as templates, followedby subcloning of the PCR products into pcDNA3.1/Hygro (InVitrogen) usingBamHI and XbaI. The primers used for construction of these variantswere: ADJ014 (see above, upstream) and:5′-GAGTCTAGATTACAGCAT-CTGGCTTCTCTT-3′ (SEQ ID NO:49) (downstream). Theresulting plasmids were termed pIGY-72 (wild-type IFNG truncated afterLeu135), pIGY-73 (S99T variant truncated after Leu135) and pIGY-74(E38N+S40T+S99T truncated after Leu135).

Generation of Cysteine-Containing IFNG Variants

INFG variants containing cysteine residues were generated usingStratagene's QuikChange™ XL site-directed mutagenesis kit, according tothe manufacturer's specifications. Seven IFNG variants, each containingone introduced cysteine, were generated using pIGY-48 as template:N10C+S99T, N16C+S99T, E38C+S99T, N59C+S99T, N83C+S99T, K94C+S99T andS99T+N104C. Similarly, six IFNG variants, each containing one introducedcysteine, were generated using pIGY-54 as template: N10C+E38N+S40T+S99T,N16C+E38N+S40T+S99T, E38N+S40T+N59C+S99T, E38N+S40T+N83C+S99T,E38N+S40T+K94C+S99T and E38N+S40T+S99T+N104C.

Example 5 PEGylation of Cysteine-Containing Variants

All buffers were de-oxidized prior to use. Protein concentrations wereestimated by measuring A280.

PEGylation Using the OPSS Coupling Chemistry

7.2 ml of 1.3 mg/ml of the IFNG variant N16C+S99T (full-length) in 5 mMsodium succinate, 4% mannitol, 0.01% Tween 20, pH 6.0, was reduced byincubation with 300 μl 0.5 M DTT for 30 minutes at room temperature. TheIFNG variant was desalted by running 3 aliquots of 2.5 ml on a NAP25 gelfiltration column (Pharmacia) in buffer A (50 mM sodium phosphate, 1 mMEDTA, pH 8.1). Each aliquote eluted in 3.5 ml.

mPEG-OPSS (10 KDa) was dissolved in buffer A to a concentration of 2mg/ml and added in equal volume to the reduced and desalted IFNG variantand incubated for 60 min with gentle shaking at room temperature.

11 ml of the reaction mixture was concentrated to 1-6 ml using aVivaspin20 column (VivaScience) and remaining mPEG was removed by gelfiltration using a Sephacryl S-100 column (Pharmacia) equilibrated inbuffer A.

The PEGylated IFNG variant was diafiltered into 5 mM sodium succinate,4% mannitol, pH 6.0 using a Vivaspin 6 column (VivaScience) and Tween 20was added to 0.01%. The purified PEGylated IFNG variant had a specificactivity of 1.3×10⁶ AU/mg as measured in the Primary Assay describedherein (15% of the specific activity of the corresponding non-PEGylatedIFNG variant).

PEGylation Using the MAL Coupling Chemistry

1.6 ml of 1.5 mg/ml of the IFNG variant N59C+S99T (full-length) in 5 mMsodium succinate, 4% mannitol, 0.01% Tween 20, pH 6.0 was reduced byincubation with 64 μl 0.5 M DTT for 30 minutes at room temperature. TheIFNG variant was desalted on a NAP25 gel filtration column (Pharmacia)in buffer A (50 mM sodium phosphate, 1 mM EDTA, pH 8.1). The INFGvariant eluted in 3.5 ml.

mPEG-MAL (5 kDa) was dissolved in buffer A to a concentration of 0.5mg/ml and added in equal volume to reduced and desalted IFNG variant andincubated for 120 minutes with gentle shaking at room temperature.

Ammonium sulphate was added to a concentration of 0.9 M and thePEGylated IFNG variant was applied onto a 1 ml Resource™ phenyl column(Pharmacia) equilibrated in buffer B (20 mM sodium phosphate, 0.9 Mammonium sulphate, pH 6.6). The column was washed with 5 column volumesof buffer B before elution of the bound PEGylated IFNG variant in alinear gradient from 0-50% buffer C (20 mM sodium phosphate, pH 6.6)over 30 column volumes. The PEGylated IFNG variant eluted around 0.6 Mammonium sulphate.

Fractions containing PEGylated IFNG variant were pooled and diafilteredinto 5 mM sodium succinate, 4% mannitol, pH 6.0 using a Vivaspin 6column (VivaScience) and Tween 20 was added to 0.01%. The purifiedPEGylated IFNG variant had a specific activity of 2.4×10⁶ AU/mg asmeasured in the Primary Assay described herein (15% of the specificactivity of the corresponding non-PEGylated IFNG variant).

Example 6 Expression of IFNG and IFNG Variants in Mammalian Cells

For transient expression of IFNG, cells were grown to 95% confluency inmedia (Dulbecco's MEM/Nut.-mix F-12 (Ham) L-glutamine, 15 mM Hepes,pyridoxine-HCl (Life Technologies Cat # 31330-038)) containing 1:10fetal bovine serum (BioWhittaker Cat # 02-701F) and 1:100 penicillin andstreptomycin (BioWhittaker Cat # 17-602E). IFNG-encoding plasmids weretransfected into the cells using Lipofectamine 2000 (Life Technologies)according to the manufacturer's specifications. 24 hrs aftertransfection, culture media were collected and assayed for IFNGactivity. Furthermore, in order to quantify the relative number ofglycosylation sites utilized, Western blotting was performed usingharvested culture medium.

Stable clones expressing IFNG were generated by transfection of CHO K1cells with IFNG-encoding plasmids followed by incubation of the cells inmedia containing 0.36 mg/ml hygromycin. Stably transfected cells wereisolated and sub-cloned by limited dilution. Clones producing highlevels of IFNG were identified by ELISA.

Example 7 Large-Scale Production

Stable cell lines expressing IFNG or variants were grown in Dulbecco'sMEM/Nut.-mix F-12 (Ham) L-glutamine, 15 mM Hepes, pyridoxine-HCl (LifeTechnologies Cat # 31330-038), 1:10 fetal bovine serum (BioWhittaker Cat# 02-701F), 1:100 penicillin and streptomycin (BioWhittaker Cat #17-602E) in 1700 cm2 roller bottles (Corning, # 431200) untilconfluence. The media was then changed to 300 ml UltraCHO withL-glutamine (BioWhittaker Cat # 12-724Q) with the addition of 1:500EX-CYTE VLE (Serological Proteins Inc. # 81-129) and 1:100 penicillinand streptomycin (BioWhittaker Cat # 17-602E). After 48 hours of growth,the media was replaced with fresh UltraCHO with the same additives.After another 48 hours of growth, the media was replaced with Dulbecco'sMEM/Nut.-mix F-12 (Ham) L-glutamine, pyridoxine-HCl (Life TechnologiesCat # 21041-025) with the addition of 1:100 ITS-A (Gibco/BRL #51300-044), 1:500 EX-CYTE VLE (Serological Proteins Inc. # 81-129) and1:100 penicillin and streptomycin (BioWhittaker Cat # 17-602E).Subsequently, every 24 h, culture media were harvested and replaced with300 ml of fresh serum-free media with the same additives. The collectedmedia were filtered through 0.22 μm filters to remove cells.

Example 8 Purification

The filtrate was microfiltrated (0.22 μm) before ultrafiltration toapproximately 1/15 volume using a Millipore TFF system. On the samesystem the concentrate was diafiltrated using 10 mM Tris, pH 7.6.Ammonium sulphate was added to a concentration of 1.7 M and afterstirring the precipitate was removed by centrifugation at 8000 rpm for25 minutes in a Sorvall centrifuge using a GS3 rotor.

The supernatant was applied onto a 25 ml Phenyl High Performance(Pharmacia) column previously equilibrated in 10 mM Tris, 1.7 M ammoniumsulphate, pH 7.6. After application the column was washed with 3 columnvolumes of 10 mM Tris, 1.7 M ammonium sulphate, pH 7.6 and the boundIFNG variant was then eluted in a linear gradient over 10 column volumesto 100% 10 mM Tris, pH 7.6. The flow-through as well as the eluted IFNGvariant was fractionated. Fractions enriched in the IFNG variant werepooled and buffer exchanged by diafiltration into 10 mM Tris, pH 9.0,using a Vivaflow200 system (VivaScience) with a molecular weight cut-offof 10,000 Da.

The IFNG variant was then applied onto a 18 ml Q-sepharose Fast Flow(Pharmacia) column previously equilibrated in 10 mM Tris, pH 9.0. Afterapplication the column was washed with 3 column volumes of 10 mM Tris,pH 9.0 before eluting the bound IFNG variant in a gradient from 0-100%10 mM Tris, 0.5 M NaCl, pH 9.0, over 15 column volumes. The flow-throughas well as the eluted IFNG variant was fractionated. Fractions enrichedin the IFNG variant were pooled and buffer exchanged into 10 mM sodiumphosphate, pH 7.0, by diafiltration using a Vivaspin20 (VivaScience)column with a molecular weight cut-off of 10,000 Da.

Then, the IFNG variant was applied onto an 8 ml CHT ceramichydroxyapatite column (Biorad) previously equilibrated in 10 mM sodiumphosphate, pH 7.0. After application the column was washed with 5 columnvolumes of 10 mM sodium phosphate, pH 7.0, before elution of the boundIFNG variant in a gradient from 0-60% 500 mM sodium phosphate, pH 7.0,over 30 column volumes. The flow-through as well as the eluted IFNGvariant was fractionated. Fractions containing the IFNG variant werepooled and buffer exchanged into 5 mM sodium succinate, 4% mannitol, pH6.0, using a VivaSpin20 column (VivaScience) and Tween 20 wassubsequently added to a concentration of 0.01%. The IFNG variant wassterile filtered and stored at −80° C.

Alternatively, the IFNG variants may be purified according to the belowpurification scheme:

The filtrate is microfiltrated (0.22 μm) before ultrafiltration toapproximately 1/15 volume using a Millipore TFF system. On the samesystem the concentrate is diafitrated using 10 mM Tris, pH 7.6, afterwhich pH is adjusted to 9.0 and precipitate is removed bymicrofiltration.

The sample is applied onto a Q-sepharose Fast Flow (Pharmacia) columnpreviously equilibrated in 10 mM Tris, pH 9.0. After application thecolumn is washed with 3 column volumes of 10 mM Tris, pH 9.0 beforeeluting the bound IFNG variant in a gradient from 0-100% 10 mM Tris, 0.5M NaCl, pH 9.0 over 15 column volumes. The flow-through as well as theeluted IFNG variant is fractionated. Fractions enriched in the INFGvariant are pooled, and pH is adjusted to 7.6. Ammonium sulphate isadded to 1.5 M and after stirring the precipitate is removed bycentrifugation.

The IFNG variant is then applied onto a Phenyl Sepharose HighPerformance (Pharmacia) previously equilibrated in 10 mM Tris, 1.5 Mammonium sulphate, pH 7.6. After application the column is washed with 3column volumes of 10 mM Tris, 1.5 M ammonium sulphate, pH 7.6, and thebound IFNG variant is then eluted in a linear gradient over 10 columnvolumes to 100% 10 mM Tris, pH 7.6. The flow-through as well as theeluted IFNG variant is fractionated. Fractions enriched in the INFGvariant are pooled and ammonium sulphate is adjusted to 1.7 M.

Then, the IFNG variant is applied onto a Butyl Sepharose columnpreviously equilibrated in 10 mM sodium phosphate, 1.7 M ammoniumsulphate, pH 7.6. After application the column is washed with 10 mMsodium phosphate, 1.7 M ammonium sulphate, pH 7.6, before eluting thebound IFNG variant in a step using 10 mM sodium phosphate, pH 6.5. Theflow-through as well as the eluted IFNG variant is fractionated.

Fractions enriched in the IFNG variant are then pooled and applied ontoa hydroxyapatite column previously equilibrated in 10 mM sodiumphosphate, pH 6.5. After application the column is washed with 5 columnvolumes of 10 mM sodium phosphate, pH 6.5, before eluting the bound IFNGvariant in a linear gradient from 0-100% 500 mM sodium phosphate, pH6.5, over 30 column volumes. The flow-through as well as the eluted IFNGvariant is fractionated.

Fractions containing the INFG variant are pooled and buffer exchangedinto a buffer containing 5 mM sodium succinate, 4% mannitol, pH 6.0.Tween 20 is subsequently added to a concentration of 0.01%. The IFNGvariant is sterile filtered and stored at −80° C.

Example 9 Activity of Variants and PEGylated Variants

Using the “primary Assay” described above, the following activity data(after transient transfection) were obtained: TABLE 1 Activity ofvariants of full-length and truncated rhuIFNG polypeptides aftertransient transfection Activity % activity of Mutations (AU/ml)full-length wt Wild-type (full-length) 1.4 × 10⁷ — S99T (full-length)5.1 × 10⁶ 36% E38N (full-length) 1.4 × 10⁷ 100%  E38N + S40T(full-length) 9.9 × 10⁶ 71% E38N + S40T + S99T (full-length) 1.3 × 10⁷93% E38N + K61T (full-length) 1.2 × 10⁷ 86% E38N + K61T + S99T(full-length) 1.4 × 10⁶ 10% N10C + S99T (full-length) 2.5 × 10⁶ 18%N16C + S99T (full-length) 1.1 × 10⁷ 79% E38C + S99T (full-length) 1.0 ×10⁷ 71% S99T + N104C (full-length) 5.0 × 10⁶ 36% N10C + E38N + S40T +S99T 1.5 × 10⁶ 11% (full-length) N16C + E38N + S40T + S99T 3.7 × 10⁶ 26%(full-length) E38N + S40T + N59C + S99T 1.1 × 10⁷ 79% (full-length)E38N + S40T + N83C + S99T 7.2 × 10⁵  5% (full-length) E38N + S40T +K94C + S99T 9.4 × 10⁵  7% (full-length) E38N + S40T + S99T + N104C 2.3 ×10⁶ 16% (full-length) Wild-type (truncated) 6.3 × 10⁶ 45% S99T(truncated) 3.5 × 10⁶ 25% E38N + S40T + S99T 4.0 × 10⁶ 29% (truncated)

Using the “primary Assay” described above, the following specificactivity data (after purification) were obtained: TABLE 2 Activity ofvariants of full-length rhuIFNG polypeptides after purification Specificactivity % activity of Mutations (AU/mg) full-length wt Wild-type(full-length) 2.1 × 10⁷ — S99T (full-length) 2.2 × 10⁷ 105%  E38N +S40T + S99T (full- 1.4 × 10⁷ 67% length) N10C + S99T (full-length) 3.8 ×10⁶ 18% N16C + S99T (full-length) 2.3 × 10⁶ 11% N16C + S99T(full-length + 1.3 × 10⁶  6% 10 kDa mPEG) E38C + S99T (full-length) 3.4× 10⁶ 16% N59C + S99T (full-length) 6.3 × 10⁶ 30% N59C + S99T(full-length + 2.4 × 10⁶ 11% 5 kDa mPEG) S99T + N104C (full-length) 3.5× 10⁶ 17%

The activity of a number of the PEGylated variants were measured bycomparing the activity of the PEGylation products by samples which havebeen subjected to the same PEGylation procedure (see Example 5 above),but without actually adding PEG to the reaction medium. The results arecompiled in Table 3 below: TABLE 3 Activity of PEGylated variants offull-length rhuIFNG polypeptides Activity relative to non-PEGylatedproduct subjected to “PEGylation procedure” Mutations (%) N10C + S99T 23(full-length + 5 kDa mPEG) N16C + S99T 59 (full-length + 5 kDa mPEG)E38C + S99T 41 (full-length + 10 kDa mPEG)¹⁾ S99T + N104C 42(full-leneth + 5 kDa mPEG)¹⁾ODSS coupling chemistry employed. MAL coupling chemistry employed forall other PEGylated variants

It should be emphasized that the activity data shown in Table 1, reflecta combination of specific activity and expression level in CHO-K1 cells.It can therefore be concluded that all variants show a comparableexpression level/specific activity to relative to rhuIFNG.

As can be seen in Table 2, all cysteine variants show a decreasedspecific activity compared to rhuIFNG while variants containing theE38N, S40T and/or S99T mutations retained a specific activity comparableto rhuIFNG. When the cysteine variants were PEGylated with either 5 or10 kDa a further 2-3 fold drop in specific activity was observed (Table3). Without being limited to any specific theory, it could be speculatedthat the decrease in specific activity could be due to decreasedreceptor binding caused by steric hindrance of the conjugated PEG group.

Example 10 Assessment of Utilization of N-Glycosylation Sites

In order to quantify the relative number of glycosylation sitesutilized, western blotting was performed using harvested culture medium(see FIG. 1). For the wild-type rhuIFNG (full-length), it was estimatedthat about 50% utilized both glycosylation sites (2N), about 40%utilized one glycosylation site (1N), and about 10% was not glycosylated(0N). These data are in agreement with previously published data byHooker et al., 1998, J. Interferon and Cytokine Res. 18, 287-295 andSarenva et al., 1995, Biochem J., 308, 9-14.

As it appears from FIG. 1, the S99T variant (full-length) utilizes itstwo glycosylation sites significantly more efficiently than thecorresponding wild-type. For the S99T variant, it was estimated thatabout 90% utilized both glycosylation sites (2N), about 7% utilized oneglycosylation site (1N), and about 3% was not glycosylated (0N).

Moreover, it is apparent from FIG. 1 that the introduced glycosylationsite at position 38 is significantly better utilized for the variantE38N+S40T (full-length) compared to the non-optimised variant E38N(full-length).

These data clearly demonstrate that better utilization of glycosylationsites, independently of whether these sites are naturally occurring orintroduced, can be achieved by introducing a threonine residue ratherthan a serine residue in position +2 relative to the asparagine residue.

Example 11 Pharmacokinetic Studies

The AUC for subcutaneous administration (AUC_(sc)) in rats wasdetermined as described hereinbefore for a number of IFNG variants. Theresults are compiled in Table 4 and in FIGS. 2 and 3. TABLE 4Pharmacokinetic data for subtunaceous administration in ratsAUC_(sc)/dose AUC_(sc,variant) AUC_(sc,variant) T_(max,sc) Variant (min× g)/ml AUC_(sc,Actimmune)® AUC_(sc,full-length, wt) (min) Actimmune ®0.3-0.4 — 0.013-0.027 43 rhuIFNG (full-length) 15-24 37-80 — 362-446E38N + S40T + S99T¹⁾ 111-192 277-640 4.6-13  308-374 N16C + S99T²⁾ 37 92-123 1.5-2.5 247 N16C + S99T³⁾ 114 285-380 4.8-7.6 249¹⁾full-length²⁾full-length, 5 kDa mPEG attached to the introduced cysteine residue³⁾full-length, 10 kDa mPEG attached to the introduced cysteine residue

Referring to FIGS. 2 and 3 and Table 4, it is evident that the variants(including PEGylated variants) have a significantly higher AUC, whenadministered subcutaneously, as compared to rhuIFNG and, in particular,when compared to the commercially available Actimmune®. Referring toFIG. 3 it should be noted that the administered dose of the twoPEGylated variants were reduced 2.5 fold compared to the administereddose of the [E38N+S40T+S99T] variant.

Evidently, this opens up the possibility of administering lower doses,thereby obtaining fewer side effects, and/or administering the activeprinciple less frequently than today thereby obtaining an improvedpatient compliance.

1-49. (canceled)
 50. A polynucleotide encoding an interferon gamma(IFNG) polypeptide S99T variant exhibiting IFNG activity and having theamino acid sequence shown in SEQ ID NO: 1, or a carboxy (C) terminallytruncated fragment thereof exhibiting IFNG activity.
 51. Apolynucleotide encoding an interferon gamma (IFNG) polypeptide S99Tvariant exhibiting IFNG activity, wherein said variant has the aminoacid sequence shown in SEQ ID NO:1.
 52. The polynucleotide according toclaim 50, wherein said encoded S99T variant is a fragment of the aminoacid sequence shown in SEQ ID NO:1 which is C-terminally truncated byremoval of 1-15 amino acid residues.
 53. The polynucleotide according toclaim 50, wherein said carboxy (C) terminally truncated fragment of saidencoded S99T variant has an amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.54. A polynucleotide encoding an interferon gamma (IFNG) polypeptideS99T variant exhibiting IFNG activity and having 1 to 10 residuemodifications relative to the amino acid sequence shown in SEQ ID NO: 1,or a carboxy (C) terminally truncated fragment thereof that exhibitsIFNG activity.
 55. The polynucleotide according to claim 54, whereinsaid encoded S99T variant comprises 1 to 10 residue modificationsrelative to the amino acid sequence shown in SEQ ID NO:
 1. 56. Thepolynucleotide according to claim 54, wherein said encoded S99T variantcomprises 1 to 10 residue modifications relative to a carboxy (C)terminally truncated fragment thereof having an amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15 and SEQ ID NO:16.
 57. The polynucleotide according to claim55, wherein said 1 to 10 residue modifications in said encoded S99Tvariant is a residue substitution.
 58. The polynucleotide according toclaim 55, wherein said encoded S99T variant comprises an amino acidresidue substitution or deletion that introduces an attachment site fora non-polypeptide moiety.
 59. The polynucleotide according to claim 58,wherein said introduced attachment site in said encoded S99T variantcomprises an introduced glycosylation site.
 60. The polynucleotideaccording to claim 59, wherein said introduced glycosylation site is anN-glycosylation site.
 61. The polynucleotide according to claim 60,wherein said N-glycosylation site is introduced by residue substitution.62. The polynucleotide according to claim 61, wherein said residuesubstitution in said encoded S99T variant is selected from the groupconsisting of K12S, K12T, G18S, G18T, E38N, E38N+S40T, K61S, K61T, N85S,N85T, K94N, Q106S and Q106T.
 63. The polynucleotide according to claim62, wherein said residue substitution in said encoded S99T variant isselected from the group consisting of K12T, G18T, E38N+S40T, K61T, N85T,K94N and Q106T.
 64. The polynucleotide according to claim 63, whereinsaid residue substitution in said encoded S99T variant is E38N+S40T. 65.The polynucleotide according to claim 58, wherein said residuesubstitution in said encoded S99T variant comprises an introducedcysteine residue.
 66. The polynucleotide according to claim 65, whereinsaid residue substitution in said encoded S99T variant is a substitutionselected from the group consisting of N10C, N16C, E38C, N59C, N83C,K94C, N104C and A124C.
 67. The polynucleotide according to claim 55,wherein said 1 to 10 residue modifications in said encoded S99T variantcomprises an introduced N-glycosylation site and an introduced cysteineresidue.
 68. The polynucleotide according to claim 67, wherein saidencoded S99T variant comprises substitutions selected from the groupconsisting of K12T+N16C, K12T+E38C, K12T+N59C, K12T+N83C, K12T+K94C,K12T+N104C, K12T+A124C, G18T+N10C, G18T+E38C, G18T+N59C, G18T+N83C,G18T+K94C, G18T+N104C, G18T+A124C, G18N+S20T+N10C, G18N+S20T+N16C,G15N+S20T+E38C, G18N+S20T+N59C, G18N+S20T+N83C, G18N+S20T+K94C,G18N+S20T+N104C, G18N+S20T+A124C, E38N+S40T+N10C, E38N+S40T+N16C,E38N+S40T+N59C, E38N+S40T+N83C, E38N+S40T+K94C, E38N+S40T+N104C,E38N+S40T+A124C, K61T+N10C, K61T+N16C, K61T+E38C, K61T+N83C, K61T+K94C,K61T+N104C, K61T+A124C, N85T+N10C, N85T+N16C, N85T+E38C, N85T+N59C,N85T+K94C, N85T+N104C, N85T+A124C, K94N+N10C, K94N+N16C, K94N+E38C,K94N+N59C, K94N+N83C, K94N+N104C, K94N+A124C, Q106T+N10C, Q106T+N16C,Q106T+E38C, Q106T+N59C, Q106T+N83C, Q106T+K94C and Q106T+A124C.
 69. Thepolynucleotide according to claim 68, wherein said encoded S99T variantcomprises substitutions selected from the group consisting ofE38N+S40T+N10C, E38N+S40T+N16C, E38N+S40T+N59C, E38N+S40T+N83C,E38N+S40T+K94C, E38N+S40T+N104C and E38N+S40T+A124C.
 70. Thepolynucleotide according to claim 55, wherein said 1 to 10 residuemodifications in said encoded S99T variant a comprises a substitution atresidue position 26 selected from the group consisting of G26F, G26N,G26Y, G26Q, G26V, G26A, G26M, G26I, G26K, G26R, G26T, G26H, G26C andG26S
 71. The polynucleotide according to claim 70, wherein saidsubstitution is selected from the group consisting of G26A, G26M, G26I,G26K, G26R, G26T, G26H, G26C and G26S
 72. The polynucleotide accordingto claim 71, wherein said substitution is selected from the groupconsisting of G26A and G26S.
 73. The polynucleotide according to claim72, wherein said substitution is G26A.
 74. The polynucleotide accordingto claim 58, wherein said encoded S99T variant comprises residuesubstitutions that remove a N-glycosylation site and introduce acysteine residue.
 75. The polynucleotide according to claim 74, whereinsaid encoded S99T variant further comprises an introducedN-glycosylation site, where said introduced N-glycosylation site isintroduced in a position different from the position occupied by saidremoved N-glycosylation site.
 76. The polynucleotide of claim 64 whereinsaid carboxy terminus of said encoded S99T variant is truncated bydeletion of 1-15 amino acid residues.
 77. The polynucleotide of claim76, wherein said carboxy terminus of said encoded S99T variant istruncated after Leu
 135. 78. The polynucleotide according to claim 52,wherein said encoded S99T variant is C-terminally truncated by removalof 11 amino acid residues.
 79. The polynucleotide according to claim 54,wherein said encoded S99T variant is C-terminally truncated by removalof 1 to 15 amino acid residues.
 80. The polynucleotide according toclaim 79, wherein said encoded S99T variant is C-terminally truncated byremoval of 11 amino acid residues.
 81. An expression vector comprisingthe polynucleotide of claim 50 operatively linked to a control element.82. An expression vector comprising the polynucleotide of claim 51operatively linked to a control element.
 83. An expression vectorcomprising the polynucleotide of claim 54 operatively linked to acontrol element.
 84. An expression vector comprising the polynucleotideof claim 64 operatively linked to a control element.
 85. An expressionvector comprising the polynucleotide of claim 80 operatively linked to acontrol element.
 86. A glycosylating host cell transformed with thepolynucleotide of claim
 50. 87. A glycosylating host cell transformedwith the polynucleotide of claim
 51. 88. A glycosylating host celltransformed with the polynucleotide of claim
 54. 89. A glycosylatinghost cell transformed with the polynucleotide of claim
 64. 90. Aglycosylating host cell transformed with the polynucleotide of claim 80.91. The glycosylating host cell of claim 86, 87, 88, 89, or 90 which isa Chinese hamster ovary (CHO) cell.
 92. The glycosylating host cell ofclaim 90, wherein the CHO cell is a CHO K1 cell.